Protein concentrates and isolates, and processes for the production thereof from macroalgae and/or microalgae

ABSTRACT

Protein concentrates and protein isolates, in addition to processes for the production of protein concentrates and protein isolates, are disclosed. In particular, the disclosure relates to the removal of fiber from macroalgae and/or microalgae using low g-force centrifugation.

PRIORITY INFORMATION

This application is claims the benefit of U.S. Provisional ApplicationNo. 61/260,148 filed on Nov. 11, 2009 and entitled PROTEIN CONCENTRATESAND ISOLATES, AND PROCESSES FOR THE PRODUCTION THEREOF FROM MICROALGAEAND/OR MACROALGAE, the contents of which of which are expresslyincorporated herein by reference.

FIELD OF THE DISCLOSURE

The present disclosure relates to protein concentrates and proteinisolates comprising combinations of proteins, peptides and amino acids,as well as processes for their production. In particular, the disclosurerelates to a process for removing fiber from macroalgae and/ormicroalgae to produce edible protein products.

BACKGROUND

Macroalgae and/or microalgae constitute a large source of availableprotein. Depending upon the identity, some macroalgae and/or microalgaecontain a high amount of fiber, as well as other antinutritional factorsand undesirable compounds, such as glucosinolates, phytic acid orphytates, sinapine and sinigrin. The fiber and antinutritional factorspresent in the protein render the macroalgae and/or microalgaeunattractive as a source of protein for commercial uses.

One method of separating the protein from the fiber, anti-nutritionalfactors and other undesirable compounds has been to dissolve themacroalgae and/or microalgae protein in a high ionic strength (i.e. highsalt content) aqueous solution. This results in the protein dissolvingin the aqueous solution, while the fiber is insoluble. However, the saltis difficult and uneconomical to remove and recover from the resultantprotein solution.

SUMMARY OF THE DISCLOSURE

Herein, a process for the production of protein concentrates and proteinisolates is disclosed. In addition, protein concentrates and proteinisolates produced in accordance with the processes of the disclosure arealso disclosed. In particular, the disclosure relates to a process forthe facile removal of fiber, antinutritional factors and otherconstituents from macroalgae and/or microalgae containing such, toproduce protein concentrates and protein isolates of high quality.

In another embodiment of the present disclosure, a process for theproduction of a protein concentrate possessing a protein content ofabout 30% to about 75% is disclosed.

Accordingly, the disclosure includes a process for the production of aprotein concentrate from macroalgae and/or microalgae, comprising:

-   -   1) removing fiber from the macroalgae and/or microalgae to form        a fiber depleted meal, comprising either:        -   i) mixing the macroalgae and/or microalgae with a mixing            solvent to form a first mixture; and            -   separating and removing fiber from the first mixture,                optionally by using a mesh screen;            -   optionally treating the mixture with phytase at a                temperature and a pH suitable for phytase activity; or        -   ii) mixing macroalgae and/or microalgae with water to form a            second mixture; and            -   optionally adjusting the pH of the second mixture to a                pH suitable for enzyme activity, optionally about 3 to                about 7, optionally 4 to 6; and            -   adding cellulase complex or other enzyme having fiber                hydrolysis activity to the second mixture and heating to                a temperature suitable for enzyme activity, to hydrolyze                the fiber;            -   optionally treating the mixture with phytase at a                temperature and a pH suitable for phytase activity,    -   2) washing the fiber depleted meal with an extraction solvent to        form an extract and a washed defatted or protein-enriched meal;    -   3) separating the extract from the washed macroalgae and/or        microalgae;    -   4) optionally repeating steps 2) and 3) at least once; and    -   5) optionally desolventizing the washed macroalgae and/or        microalgae to form a protein concentrate.

In another embodiment, the mixing solvent comprises water, methanol,ethanol or isopropanol, and mixtures thereof. In a further embodiment,the solvent is water or ethanol, and mixtures thereof. In an embodimentof the disclosure, the macroalgae and/or microalgae is mixed with amixing solvent in a ratio of about 3 to about 10 parts solvent to about1 part of the macroalgae and/or microalgae, optionally about 4 to about8, or about 4 to about 6, on a weight-to-weight basis.

In another embodiment of the disclosure, the mixture is screened througha mesh screen of typically about 10 to about 200 US mesh size,optionally a mesh screen of about 20 to about 200 US mesh size. Inanother embodiment, the mesh size is 40 US mesh size.

In an embodiment of the present disclosure, the macroalgae and/ormicroalgae is mixed thoroughly with water to form the second mixture. Inan embodiment, the mixing of water and the macroalgae and/or microalgaecomprises using a wet mill or an inline mixer.

In another embodiment of the present disclosure, the cellulase complexis added to the second mixture in an amount of about 1 gram to about 10grams for about every 1 kg of dry solids of the macroalgae and/ormicroalgae (about 0.1% to about 1%). In a further embodiment, thecellulase complex is mixed with the second mixture for about 0.5 hoursto about 5 hours. In another embodiment, the cellulase complex is mixedwith the second mixture for about 1 to about 3 hours.

In another embodiment of the disclosure, the second mixture with theadded cellulase complex is heated to a temperature of about 30° C. toabout 60° C., suitably about 40° C. to about 60° C.

In an embodiment, the cellulase complex comprises at least one ofendocellulase, exocellulase, cellobiohydrolase, cellobiase,endohemicellulase and exohemicellulase.

In an embodiment of the disclosure, the extraction solvent comprisesmethanol, ethanol or isopropanol, and mixtures thereof. In a furtherembodiment, the extraction solvent comprises ethanol or water, andmixtures thereof.

In an embodiment of the present disclosure, the first or second mixtureis washed at least once with about 5% to about 100%, optionally about25% to about 85%, or about 50% to about 85%, or about 60% to about 85%,of the extraction solvent (v/v) in water.

In an embodiment of the present disclosure, the ratio of the extractionsolvent to the first or second mixture is about 5% to about 95%,optionally about 10% to about 90%, about 20% to about 70%, or about 40%to about 80% (v/v) (extraction solvent to first or second mixture).

In an embodiment of the present disclosure, the first or second mixtureis washed with the extraction solvent at a temperature of about 10° C.to about 90° C. In another embodiment, the first or second mixture iswashed with the extraction solvent at a temperature of about 20° C. toabout 60° C. In a further embodiment, the first or second mixture iswashed with the extraction solvent at a temperature of about 20° C. toabout 25° C.

In another embodiment of the present disclosure, the extract isseparated from the washed macroalgae and/or microalgae bycentrifugation, vacuum filtration, pressure filtration, decantation orgravity draining in an extractor.

In another embodiment of the present disclosure, steps 2) and 3) arerepeated at least twice.

In another embodiment of the present disclosure, the process furthercomprises the step of drying the washed macroalgae and/or microalgae toform the protein concentrate. In a further embodiment, the washedmacroalgae and/or microalgae is dried in a vacuum dryer, fluidized beddryer, ring dryer or spray dryer. In another embodiment, the washedmacroalgae and/or microalgae is dried to a moisture content of about0.5% to about 12%, optionally about 1% to about 10%, about 4% to about8%. In a further embodiment, the washed macroalgae and/or microalgae isdried to a moisture content of about 6%.

In another embodiment of the present disclosure, the extract isdesolventized and dried to form a high sugar fraction. In an embodiment,the extract is desolventized by spray drying, drum drying or vacuumdrying.

In an embodiment of the present disclosure, a process for the productionof a protein concentrate possessing a protein content of about 75% toabout 90% is disclosed. In another embodiment, the protein concentrateis hydrolyzed to produce peptides and free amino acids. In anotherembodiment, the hydrolyzed protein concentrate comprises peptides and/orfree amino acids.

The disclosure also includes a process for the production of a proteinconcentrate from macroalgae and/or microalgae, comprising:

removing fiber from the macroalgae and/or microalgae, comprising:

-   -   i) mixing the macroalgae and/or microalgae with a mixing solvent        to form a mixture;        -   separating fiber from the mixture, optionally by screening            the mixture to remove fiber,        -   optionally adjusting the pH of the mixture to a pH of about            4.5 to about 8.0, optionally about 6.5 to about 7.5, or            optionally about 7;        -   optionally treating the mixture with phytase at a            temperature and a pH suitable for phytase activity,        -   optionally milling the mixture;        -   separating fiber, optionally by centrifuging the mixture, to            remove fiber,        -   thereby forming a protein slurry; and    -   ii) separating the protein slurry, optionally by centrifuging        the protein slurry, to form a protein precipitate and a soluble        protein fraction;    -   iii) washing the protein precipitate with an extraction solvent        at least once and separating, optionally by centrifuging, to        form a purified protein precipitate;    -   iv) optionally drying the purified protein precipitate to form        the protein concentrate.

In another embodiment of the disclosure, the mixing solvent compriseswater, methanol, ethanol, or isopropanol, and mixtures thereof. In afurther embodiment, the mixing solvent comprises water or ethanol, andmixtures thereof. In another embodiment, the ratio of macroalgae and/ormicroalgae to the mixing solvent is about 1:3 to about 1:20. In afurther embodiment, the ratio is about 1:6 to about 1:10. In anembodiment, the ratio is about 1:6 to about 1:8.

In another embodiment of the disclosure, the mixture is screened througha mesh screen of typically about 10 to about 200 US mesh size,optionally a mesh screen of about 20 to about 200 US mesh size. Inanother embodiment, the mesh size is 40 US mesh size.

In another embodiment of the present disclosure, the pH of mixture isadjusted with aqueous sodium hydroxide. In an embodiment, the aqueoussodium hydroxide has a concentration of about 1% to about 40% by weightof sodium hydroxide. In a further embodiment, the concentration ofsodium hydroxide is about 5% to about 30% sodium hydroxide.

In another embodiment, the optional milling step comprises using a wetmill.

In an embodiment, the mixture is centrifuged using a decantingcentrifuge. In an embodiment, the mixture is centrifuged with adecanting centrifuge at a speed of about 500 rpm to about 6000 rpm. Inanother embodiment, the speed is about 1500 rpm.

In an embodiment of the disclosure, the protein slurry is centrifugedusing a decanter or disc stack centrifuge. In a further embodiment, theprotein slurry is centrifuged at a speed of about 2500 rpm to about 8500rpm.

In another embodiment of the disclosure, the extraction solvent iswater, methanol, ethanol or isopropanol, and mixtures thereof. In afurther embodiment, the extraction solvent is water or ethanol, andmixtures thereof. In an embodiment, the extraction solvent is water. Inan embodiment, the protein precipitate is washed at least twice with theextraction solvent.

In an embodiment of the present disclosure, the washed proteinprecipitate is centrifuged with a disc stack centrifuge at a speed ofabout 7500 rpm to about 8500 rpm.

In an embodiment of the disclosure, the purified protein precipitate isdried in a vacuum dryer, fluidized bed dryer, ring dryer or spray dryerto form the protein concentrate. In a further embodiment, the proteinconcentrate is dried to a moisture content of about 1% to about 10%. Inanother embodiment, the protein concentrate is dried to a moisturecontent of about 6%.

In another embodiment, the protein concentrate comprises a hydrolyzedprotein concentrate. In another embodiment, the protein concentrate ishydrolyzed to produce peptides and free amino acids. In anotherembodiment, the hydrolyzed protein concentrate comprises peptides and/orfree amino acids.

In another embodiment of the present disclosure, a process for theproduction of a protein isolate possessing a protein content of greaterthan about 90% is disclosed.

Accordingly, the disclosure includes a process for the production of aprotein isolate from macroalgae and/or microalgae, comprising:

removing fiber from the macroalgae and/or microalgae, comprising:

-   -   i) mixing the macroalgae and/or microalgae with a mixing solvent        to form a mixture;        -   separating fiber from the mixture to remove fiber,        -   optionally adjusting the pH of the mixture to a pH of about            6.0 to about 8.0, optionally about 6.5 to about 7.5, or            optionally about 7;        -   optionally milling the mixture;        -   optionally treating the mixture with phytase at a            temperature and a pH suitable for phytase activity,        -   separating fiber, optionally by centrifuging the mixture, to            remove fiber,

thereby forming a protein slurry;

-   -   ii) separating the protein slurry, optionally by centrifuging        the protein slurry, to form a protein precipitate and a soluble        protein fraction;    -   iii) filtering the soluble protein fraction to separate it from        protein precipitate; and    -   iv) optionally drying the soluble protein to form the protein        isolate.

In another embodiment of the disclosure, the mixing solvent compriseswater or a salt solution. In an embodiment, the salt solution comprisesless than 5%, optionally about 3% to about 4%, or 3.5% by weight of saltin solution. In a further embodiment, the mixing solvent compriseswater. In another embodiment, the ratio of macroalgae and/or microalgaeto the mixing solvent is about 1:3 to about 1:20. In a furtherembodiment, the ratio is about 1:6 to about 1:10. In an embodiment, theratio is about 1:6 to about 1:8.

In another embodiment of the present disclosure, the pH of mixture isadjusted with aqueous sodium hydroxide. In an embodiment, the aqueoussodium hydroxide has a concentration of about 1% to about 40% by weightof sodium hydroxide. In a further embodiment, the concentration ofsodium hydroxide is about 5% to about 30% sodium hydroxide.

In another embodiment of the disclosure, the mixture is screened througha mesh screen of about 10 to about 200 US mesh size, optionally a meshscreen of about 20 to about 200 US mesh size. In an embodiment, the meshsize is 40 US mesh size.

In an embodiment, the mixture is centrifuged using a decantingcentrifuge. In an embodiment, the mixture is centrifuged with adecanting centrifuge at a speed of about 500 rpm to about 6000 rpm. Inanother embodiment, the speed is about 1500 rpm.

In another embodiment, the optional milling step comprises using a wetmill.

In an embodiment of the disclosure, the protein slurry is centrifugedusing a disc stack centrifuge. In a further embodiment, the proteinslurry is centrifuged at a speed of about 6500 rpm to about 8500 rpm.

In another embodiment of the disclosure, the soluble protein fraction isfiltered using an ultrafiltration or diafiltration apparatus. In afurther embodiment, the ultrafiltration or diafiltration apparatuscomprises a membrane to filter proteins of larger than about 1,000daltons, optionally 10,000 daltons, optionally about 30,000 daltons, orabout 100,000 daltons. In another embodiment, the ultrafiltration ordiafiltration is performed at a temperature of about 1° C. to about 60°C., optionally about 40° C. to about 55° C.

In another embodiment of the disclosure, the soluble protein is dried ina vacuum dryer, fluidized bed dryer, ring dryer or spray dryer to formthe protein isolate. In an embodiment, the protein isolate is dried to amoisture content of about 1% to about 10%. In a further embodiment, theprotein isolate is dried to a moisture content of about 6%.

In another embodiment, the protein isolate comprises a hydrolyzedprotein isolate. In another embodiment, the protein isolate ishydrolyzed to produce peptides and free amino acids. In anotherembodiment, the hydrolyzed protein isolate comprises peptides and/orfree amino acids.

In another embodiment of the present disclosure, a process for theproduction of a protein isolate possessing a protein content of greaterthan about 90% is disclosed.

Accordingly, the disclosure includes a process for the production of aprotein isolate from macroalgae and/or microalgae, comprising:

removing fiber from the defatted or protein-enriched algae, comprising:

-   -   i) mixing the macroalgae and/or microalgae with a mixing solvent        to form a mixture;        -   separating fiber from the mixture to remove fiber,        -   optionally adjusting the pH of the mixture to a pH of about            6.0 to about 8.0, optionally about 6.5 to about 7.5, or            optionally about 7;        -   optionally milling the mixture;        -   optionally treating the mixture with phytase at a            temperature and a pH suitable for phytase activity,        -   separating fiber, optionally by centrifuging the mixture, to            remove fiber,        -   thereby forming a protein slurry; and    -   ii) separating the protein slurry, optionally by centrifuging        the protein slurry, to form a protein precipitate and a soluble        protein fraction;    -   iii) mixing the protein precipitate with water to form a protein        precipitate mixture and optionally adjusting the pH to a pH        suitable for enzyme activity, optionally about 3 to about 7,        optionally about 4 to about 6;    -   iv) adding cellulase complex or other enzyme having fiber        hydrolysis activity to the protein precipitate mixture to        hydrolyze fiber, typically residual fiber;    -   v) washing the protein precipitate mixture with an extraction        solvent at least once and separating, optionally by        centrifuging, to form a protein isolate.

In another embodiment of the disclosure, the mixing solvent compriseswater or a salt solution. In an embodiment, the salt solution comprisesless than 5%, optionally about 3% to about 4%, or 3.5% by weight of saltin solution. In a further embodiment, the mixing solvent compriseswater. In another embodiment, the ratio of macroalgae and/or microalgaeto the mixing solvent is about 1:3 to about 1:20. In a furtherembodiment, the ratio is about 1:6 to about 1:10. In an embodiment, theratio is about 1:6 to about 1:8.

In another embodiment of the disclosure, the mixture is screened througha mesh screen of about 10 to about 200 US mesh size, optionally a meshscreen of about 20 to about 200 US mesh size. In another embodiment, themesh size is 40 US mesh size.

In another embodiment of the present disclosure, the pH of mixture isadjusted with aqueous sodium hydroxide. In an embodiment, the aqueoussodium hydroxide has a concentration of about 1% to about 40% by weightof sodium hydroxide. In a further embodiment, the concentration ofsodium hydroxide is about 5% to about 30% sodium hydroxide.

In another embodiment, the optional milling step comprises using a wetmill.

In an embodiment, the mixture is centrifuged using a decantingcentrifuge. In an embodiment, the mixture is centrifuged with adecanting centrifuge at a speed of about 500 rpm to about 6000 rpm. Inanother embodiment, the speed is about 1500 rpm.

In an embodiment of the disclosure, the protein slurry is centrifugedusing a disc stack centrifuge. In a further embodiment, the proteinslurry is centrifuged at a speed of about 6500 rpm to about 8500 rpm.

In another embodiment of the disclosure, the cellulase complex is addedto the protein precipitate mixture in an amount of about 0.1% to about1% by weight of the protein precipitate mixture. In a furtherembodiment, the cellulase complex is mixed with the protein precipitatemixture for about 0.5 hours to about 5 hours. In another embodiment, thecellulase complex is mixed with the protein precipitate mixture forabout 1 to about 3 hours. In a further embodiment, the cellulase complexcomprises at least one of endocellulase, exocellulase,cellobiohydrolase, cellobiase, endohemicellulase and exohemicellulase.In an embodiment, the protein precipitate mixture with cellulase complexis heated to a temperature of about 30° C. to about 60° C., optionallyabout 40° C. to about 60° C.

In another embodiment of the disclosure, the mixing solvent compriseswater. In another embodiment, the ratio of macroalgae and/or microalgaeto the mixing solvent is about 1:3 to about 1:20. In a furtherembodiment, the ratio is about 1:6 to about 1:10. In an embodiment, theratio is about 1:6 to about 1:8.

In another embodiment of the present disclosure, the protein precipitatemixture is centrifuged using a decanter or disc stack centrifuge. In afurther embodiment, the protein precipitate mixture is centrifuged at aspeed of about 2500 rpm to about 8500 rpm.

In another embodiment of the present disclosure, the protein isolate issubjected to high pressure jet cooking.

In an embodiment of the present disclosure, the protein isolate ishydrolyzed using proteases to form a hydrolyzed protein extract. In afurther embodiment, the proteases comprise Alcalase® (serineendopeptidase, typically from Bacillus subtilis), or Flavourzyme®(fungal protease/peptidase complex, typically produced from Aspergillusoryzae fermentation), both proteases from Novozymes® North America, Inc.In an embodiment, the ratio of Alcalase® to the protein isolate is about0.1% to about 1%. In another embodiment, the ratio of Alcalase® to theprotein isolate is about 0.5%. In a further embodiment, the ratio ofFlavourzyme® to the protein isolate is about 0.1% to about 1%. In anembodiment, the ratio of Flavourzyme® to the protein isolate is about0.5%.

In another embodiment, the protein isolate comprises a hydrolyzedprotein isolate. In another embodiment, the protein isolate ishydrolyzed to produce peptides and free amino acids. In anotherembodiment, the hydrolyzed protein isolate comprises peptides and/orfree amino acids.

In another embodiment of the disclosure, there is a provided a processfor the production of a protein concentrate from macroalgae and/ormicroalgae, comprising:

-   -   i) mixing the macroalgae and/or microalgae with a mixing solvent        to form a mixture and optionally treating the mixture with        phytase at a temperature and a pH suitable for phytase activity,    -   ii) optionally adjusting the pH of the mixture to a pH of about        2.0 to about 10.0;    -   iii) separating fiber from the mixture to form a protein slurry,        wherein the protein slurry comprises a soluble protein fraction        and an insoluble protein fraction;    -   iv) optionally repeating steps i)-iii) by mixing the protein        slurry with additional partially defatted, fully defatted or        protein-enriched algae;    -   v) mixing the protein slurry with an extraction solvent to form        an extract and a washed insoluble protein fraction;    -   vi) separating the extract from the washed insoluble protein        fraction;    -   vii) optionally repeating steps v) and vi) at least once; and    -   viii) optionally desolventizing the washed insoluble protein        fraction to form a protein concentrate.

In another embodiment of the disclosure, the ratio of macroalgae and/ormicroalgae to mixing solvent is about 1:3 to about 1:30 (w/w).

In another embodiment, the ratio of macroalgae and/or microalgae tosolvent is about 1:5 to about 1:20 (w/w). In a further embodiment, theratio is about 1:6 to about 1:12 (w/w). In an embodiment, the ratio isabout 1:8 to about 1:10 (w/w).

In a further embodiment of the disclosure, the mixing solvent compriseswater or an aqueous solution comprising a polysaccharide, a salt, suchas sodium chloride, potassium chloride, or calcium chloride, or analcohol. In an embodiment, the mixing solvent is water. In anotherembodiment, the polysaccharide is guar gum.

In an embodiment, the pH of the protein slurry is adjusted to a pH ofabout 6.5 to about 10.0. In a further embodiment, the pH of the proteinslurry is adjusted to a pH of about 7.0 to about 9.0.

In another embodiment of the disclosure, the mixture is separated bycentrifugation or hydrocyclone to separate the fiber from the mixtureand form the protein slurry. In a further embodiment, the mixture isseparated by centrifugation to separate the fiber from the mixture andform the protein slurry. In an embodiment, the mixture is centrifuged ata speed of about 1,000 rpm to about 2,000 rpm. In a further embodiment,the mixture is centrifuged at a speed of about 1,400 to about 1,600 rpm.In an embodiment, the mixture is centrifuged using a decantercentrifuge.

In another embodiment of the disclosure, mixing the protein slurry withadditional macroalgae and/or microalgae is repeated at least once. In afurther embodiment, mixing the protein slurry with additional macroalgaeand/or microalgae is repeated at least two to seven times.

In an embodiment of the disclosure, the extraction solvent compriseswater, methanol, ethanol, isopropanol, or mixtures thereof. In anembodiment, the extraction solvent comprises ethanol. In anotherembodiment, the extraction solvent comprises at least about 50% ethanol.In an embodiment, the extraction solvent comprises at least about 70%ethanol. In a further embodiment, the extraction solvent comprises atleast about 90% ethanol.

In a further embodiment, the extract is separated from the washedinsoluble protein fraction using centrifugation, vacuum filtration,pressure filtration, decantation or gravity draining. In an embodiment,the extract is separated from the washed insoluble protein fractionusing centrifugation.

In another embodiment of the disclosure, wherein steps iv) and v) arerepeated at least twice.

In a further embodiment, the process further comprises the step ofdrying the washed insoluble protein fraction to form the proteinconcentrate. In an embodiment, the protein concentrate is dried in avacuum dryer, fluidized bed dryer, hot air dryer ring dryer or spraydryer.

In another embodiment, the protein concentrate comprises a hydrolyzedprotein concentrate. In another embodiment, the protein concentrate ishydrolyzed to produce peptides and free amino acids. In anotherembodiment, the hydrolyzed protein concentrate comprises peptides and/orfree amino acids.

In an embodiment, the protein concentrate comprises a protein content ofabout 30% to about 90% on a dry weight basis.

In another embodiment of the disclosure, there is also provided aprocess for the production of a protein isolate from macroalgae and/ormicroalgae, comprising:

-   -   i) mixing the macroalgae and/or microalgae with a blending        solvent, optionally water or alkaline water, to form a mixture        and optionally treating the mixture with phytase at a        temperature and a pH suitable for phytase activity;    -   ii) optionally adjusting the pH of the mixture to a pH of about        7.0 to about 10.0;    -   iii) separating fiber from the mixture to form a first protein        slurry, wherein the first protein slurry comprises a soluble        protein fraction and an insoluble protein fraction;    -   iv) separating the first protein slurry to form a protein solids        fraction and a soluble protein fraction;    -   v) optionally mixing the protein solids fraction with a second        blending solvent, optionally water, to form a second protein        slurry;    -   vi) optionally separating the second protein slurry to form a        second protein solids fraction and a second soluble protein        fraction;    -   vii) optionally repeating steps v) and vi) at least once;    -   viii) separating the soluble protein fractions to form a        clarified soluble protein fraction and a residual insoluble        protein fraction;    -   ix) optionally adjusting the pH of the clarified soluble protein        fraction to a pH of about 6 to about 9;    -   x) separating the clarified soluble protein fraction, optionally        by filtering the clarified soluble protein fraction by membrane        filtration; and    -   xi) optionally drying the clarified soluble protein fraction.

In another embodiment of the disclosure, the ratio of macroalgae and/ormicroalgae to water or alkaline water is about 1:4 to about 1:30 (w/w).In another embodiment, the ratio of macroalgae and/or microalgae towater or alkaline water is about 1:5 to about 1:20 (w/w). In a furtherembodiment, the ratio is about 1:6 to about 1:12 (w/w). In anembodiment, the ratio is about 1:8 to about 1:10 (w/w).

In an embodiment of the disclosure, the pH of the alkaline water isabout 7 to about 12. In another embodiment, the pH of the first proteinslurry is adjusted to about 8.0 to about 9.5. In a further embodiment,the pH of the first protein slurry is adjusted to about 8.5 to about9.0.

In another embodiment of the disclosure, the mixture is separated bycentrifugation or hydrocyclone to separate the fiber from the mixtureand form the protein slurry. In a further embodiment, the mixture isseparated by centrifugation to separate the fiber from the mixture andform the protein slurry. In an embodiment, the mixture is centrifuged ata speed of about 1,000 rpm to about 2,000 rpm. In a further embodiment,the mixture is centrifuged at a speed of about 1,400 to about 1,600 rpm.In an embodiment, the mixture is centrifuged using a decantercentrifuge.

In another embodiment, the first protein slurry is centrifuged,optionally using a disc stack centrifuge, to separate the protein solidsfraction from the soluble protein fraction. In a further embodiment, thefirst protein slurry is centrifuged at a speed of about 4,000 rpm toabout 8,000 rpm. In a further embodiment, the first protein slurry iscentrifuged at a speed of about 6,500 to about 7,500 rpm.

In another embodiment of the disclosure, the ratio of the protein solidsfraction to water is about 1.0:0.5 to about 1.0:3.0 (w/w). In a furtherembodiment, the ratio of the protein solids fraction to water is about1.0:1.0 to about 1.0:2.0 (w/w).

In an embodiment, the soluble protein fractions are centrifuged to formthe clarified soluble protein fraction and the residual insolubleprotein fraction. In an embodiment, the soluble protein fractions arecentrifuged using a disc stack centrifuge at a speed of about 7,000 rpmto about 10,000 rpm. In a further embodiment, the soluble proteinfractions are centrifuged using a disc stack centrifuge at a speed ofabout 7,500 rpm to about 8,500 rpm.

In another embodiment of the disclosure, the pH of the clarified solubleprotein fraction is adjusted with alkali. In a further embodiment, thepH of the clarified soluble protein fraction is adjusted with sodiumhydroxide.

In an embodiment, the clarified soluble protein fraction is filteredusing an ultrafiltration apparatus. In a further embodiment, theultrafiltration apparatus comprises a membrane to filter proteins largerthan about 10,000 daltons.

In another embodiment of the disclosure, the process further comprisesthe step of filtering the clarified soluble protein fraction using adiafiltration apparatus.

In another embodiment, the clarified soluble protein fraction is driedin a vacuum dryer, fluidized bed dryer, ring dryer or spray dryer toform the protein isolate.

In another embodiment, the protein isolate comprises a hydrolyzedprotein isolate. In another embodiment, the protein isolate ishydrolyzed to produce peptides and free amino acids. In anotherembodiment, the hydrolyzed protein isolate comprises peptides and/orfree amino acids.

In another embodiment of the disclosure, the protein isolate comprises aprotein content of greater than about 90% on a dry weight basis.

In another embodiment of the disclosure, there is also provided aprocess for the production of a hydrolyzed protein concentrate frommacroalgae and/or microalgae, comprising:

-   -   i) mixing the macroalgae and/or microalgae with a blending        solvent, optionally water, to form a first mixture and        optionally treating the mixture with phytase at a temperature        and a pH suitable for phytase activity;    -   ii) optionally adjusting the pH of the first mixture to a pH of        about 6.5 to about 10.0;    -   iii) separating the first mixture to remove fiber from the first        mixture and form a protein slurry and an insoluble fiber        fraction, wherein the protein slurry comprises a soluble protein        fraction and an insoluble protein fraction and the insoluble        fiber fraction comprises insoluble fiber and a second insoluble        protein fraction;    -   iv) optionally mixing the insoluble fiber fraction with a second        blending solvent, optionally water, to form a washed insoluble        fiber fraction and an extract;    -   v) separating the washed insoluble fiber fraction from the        extract;    -   vi) optionally mixing the washed insoluble fiber fraction with a        blending solvent, optionally water, to form a second mixture;    -   vii) optionally adjusting the pH of the second mixture to a pH        suitable for enzymatic activity;    -   viii) mixing the second mixture with at least one protease to        form a hydrolyzed protein extract;    -   ix) separating the hydrolyzed protein extract from the second        mixture to form the hydrolyzed protein concentrate and a second        insoluble fiber fraction; and    -   x) optionally drying the hydrolyzed protein concentrate.

In another embodiment of the disclosure, the ratio of macroalgae and/ormicroalgae to water is about 1:4 to about 1:30 (w/w). In anotherembodiment, the ratio of macroalgae and/or microalgae to water is about1:5 to about 1:20 (w/w). In a further embodiment, the ratio is about 1:6to about 1:12 (w/w). In an embodiment, the ratio is about 1:8 to about1:10 (w/w).

In another embodiment, the pH of the first mixture is adjusted to about8.0 to about 9.5. In a further embodiment, the pH of the first mixtureis adjusted to about 8.5 to about 9.0.

In another embodiment of the disclosure, the first mixture is separatedby centrifugation or hydrocyclone to separate the fiber from the firstmixture and form the protein slurry. In a further embodiment, themixture is separated by centrifugation to separate the fiber from themixture and form the protein slurry. In an embodiment, the first mixtureis centrifuged at a speed of about 1,000 rpm to about 2,000 rpm. In afurther embodiment, the first mixture is centrifuged at a speed of about1,400 to about 1,600 rpm. In an embodiment, the mixture is centrifugedusing a decanter centrifuge.

In another embodiment, the ratio of the insoluble fiber fraction orwashed insoluble fiber fraction to water is about 1.0:0.5 to about1.0:3.0 (w/w). In a further embodiment, the ratio of the insoluble fiberfraction or washed insoluble fiber fraction to water is about 1.0:1.0 toabout 1.0:2.0 (w/w).

In another embodiment, the washed insoluble fiber fraction iscentrifuged to separate the washed insoluble fiber fraction fromextract. In a further embodiment, the washed insoluble fiber fraction iscentrifuged at a speed of about 2,000 rpm to about 6,000 rpm. In afurther embodiment, washed insoluble fiber fraction is centrifuged at aspeed of about 3,000 to about 5,500 rpm.

In another embodiment of the disclosure, the pH of the second mixture isadjusted to about 8.0 to about 9.0.

In an embodiment of the disclosure, the ratio of the second mixture tothe protease is about 100:1 to about 5000:1 (w/w).

In an embodiment of the disclosure, the second mixture is mixed with aprotease at a temperature of about 40° C. to about 60° C. In anotherembodiment, the second mixture is mixed with a protease at a temperatureof about 45° C. to about 55° C.

In another embodiment, the at least one protease comprises a proteasefrom Bacillus Licheniformis.

In a further embodiment, the process further comprises the step ofmixing the second mixture with a second protease.

In an embodiment, the ratio of the second mixture to the second proteaseis about 250:1 to about 5000:1 (w/w).

In another embodiment, the second mixture is mixed with the secondprotease at a temperature of about 50° C. to about 70° C. In anembodiment, the second mixture is mixed with the second protease at atemperature of about 55° C. to about 65° C.

In a further embodiment, the second protease comprises a fungalprotease/peptidase complex from Aspergillus oryzae.

In another embodiment, the hydrolyzed protein concentrate is dried in avacuum dryer, fluidized bed dryer, ring dryer or spray dryer to form theprotein isolate.

In a further embodiment, the hydrolyzed protein concentrate comprises aprotein content of about 30% to about 90% on a dry weight basis.

In another embodiment, the process further comprises mixing thehydrolyzed protein extract with water to form a third mixture. In afurther embodiment, the process further comprises filtering the thirdmixture fraction and the filtering comprises ultrafiltration. In anembodiment, the ultrafiltration comprises contacting the third mixturewith an ultrafiltration apparatus that comprises a membrane to filterproteins larger than about 1,000 daltons.

In another embodiment, the process further comprises mixing the secondinsoluble fiber fraction to form a washed hydrolyzed protein extract anda washed second insoluble fiber fraction and separating the form thewashed hydrolyzed protein extract from the washed second insoluble fiberfraction. In another embodiment, the washed hydrolyzed protein extractis combined with the hydrolyzed protein extract.

In an embodiment of the disclosure, there is also provided a process forthe production of a protein concentrate from macroalgae and/ormicroalgae comprising:

-   -   i) mixing the macroalgae and/or microalgae with a blending        solvent, optionally water, a saline solution or a polysaccharide        solution, to form a mixture and optionally treating the mixture        with phytase at a temperature and a pH suitable for phytase        activity;    -   ii) optionally adjusting the pH of the mixture to a pH of about        2.0 to about 10.0;    -   iii) separating fiber from the mixture to form a protein slurry,        wherein the protein slurry comprises a first soluble protein        fraction and an insoluble protein fraction;    -   iv) optionally repeating steps i)-iii) by mixing the protein        slurry with additional algae;    -   v) separating the soluble protein fraction from the insoluble        protein fraction;    -   vi) washing the insoluble protein fraction with a second        blending solvent, optionally water, saline solution or        polysaccharide solution, to form a washed insoluble protein        fraction and a second soluble protein fraction;    -   vii) separating the washed insoluble protein fraction and the        second soluble protein fraction;    -   viii) combining and separating the first and second soluble        protein fractions to form a protein concentrate, optionally by        filtering the first and second soluble protein fractions to form        a protein concentrate or isolate;    -   ix) combining the washed insoluble protein fraction with the        protein concentrate to form a combined protein concentrate or        isolate; and    -   x) optionally drying the combined protein concentrate.

In another embodiment of the disclosure, the ratio of macroalgae and/ormicroalgae to water is about 1:3 to about 1:30 (w/w). In anotherembodiment, the ratio of macroalgae and/or microalgae to water is about1:5 to about 1:20 (w/w). In a further embodiment, the ratio is about 1:6to about 1:12 (w/w). In an embodiment, the ratio is about 1:8 to about1:10 (w/w).

In an embodiment, the pH of the mixture is adjusted to a pH of about 6.5to about 10.0. In another embodiment, the pH of the mixture is adjustedto a pH of about 7.0 to about 9.0.

In another embodiment of the disclosure, the mixture is separated bycentrifugation or hydrocyclone to separate the fiber from the mixtureand form the protein slurry. In a further embodiment, the mixture isseparated by centrifugation to separate the fiber from the mixture andform the protein slurry. In an embodiment, the mixture is centrifuged ata speed of about 1,000 rpm to about 2,000 rpm. In a further embodiment,the mixture is centrifuged at a speed of about 1,400 to about 1,600 rpm.In an embodiment, the mixture is centrifuged using a decantercentrifuge.

In another embodiment, the protein slurry is centrifuged to separate theprotein solids fraction from the soluble protein fraction. In anembodiment, the protein slurry is centrifuged at a speed of about 6,000rpm to about 8,500 rpm in a disc stack centrifuge. In anotherembodiment, the protein slurry is centrifuged at a speed of about 6,500to about 7,500 rpm.

In another embodiment, the ratio of the insoluble protein fraction towater is about 1.0:0.5 to about 1.0:3.0 (w/w). In a further embodiment,the ratio of the insoluble protein fraction to water is about 1.0:1.0 toabout 1.0:2.0 (w/w).

In another embodiment, the washed insoluble protein fraction and thesecond soluble protein fraction are separated using a centrifuge. In anembodiment, the washed insoluble protein fraction and the second solubleprotein fraction are centrifuged at a speed of about 6,000 rpm to about8,500 rpm in a disc stack centrifuge. In a further embodiment, thewashed insoluble protein fraction and the second soluble proteinfraction are centrifuged at a speed of about 6,500 to about 7,500 rpm.

In another embodiment, the first and second soluble protein fractionsare filtered using an ultrafiltration apparatus. In a furtherembodiment, the ultrafiltration apparatus comprises a membrane to filterproteins larger than about 10,000 daltons. In an embodiment, the processfurther comprises the step of filtering the first and second solubleprotein fractions using a diafiltration apparatus.

In another embodiment, the combined protein concentrate is dried in avacuum dryer, fluidized bed dryer, ring dryer or spray dryer to form thedried protein concentrate.

In another embodiment, the protein concentrate comprises a hydrolyzedprotein concentrate. In another embodiment, the protein concentrate ishydrolyzed to produce peptides and free amino acids. In anotherembodiment, the hydrolyzed protein concentrate comprises peptides and/orfree amino acids.

In a further embodiment, the protein concentrate comprises a proteincontent of about 50% to about 90% on a dry weight basis.

In an embodiment of the disclosure, there is also provided a process forthe production of a protein isolate from macroalgae and/or microalgaecomprising:

-   -   i) mixing the macroalgae and/or microalgae with a blending        solvent, optionally water, to form a mixture and optionally        treating the mixture with phytase at a temperature and a pH        suitable for phytase activity;    -   ii) optionally adjusting the pH of the mixture to a pH of about        2.0 to about 10.0;    -   iii) separating fiber from the mixture to form a protein slurry,        wherein the protein slurry comprises a soluble protein fraction        and an insoluble protein fraction;    -   iv) washing the fiber with a second blending solvent, optionally        water, to form a washed fiber fraction;    -   vi) separating the washed fiber fraction to form a second        protein slurry and washed fiber solids;    -   vii) combining and separating the first and second protein        slurries to form a protein concentrate, optionally by filtering        the first and second soluble protein fractions to form a protein        concentrate; and    -   ix) optionally drying the protein concentrate.

In another embodiment of the disclosure, the ratio of macroalgae and/ormicroalgae to water is about 1:3 to about 1:30 (w/w). In anotherembodiment, the ratio of macroalgae and/or microalgae to water is about1:5 to about 1:20 (w/w). In a further embodiment, the ratio is about 1:6to about 1:12 (w/w). In an embodiment, the ratio is about 1:8 to about1:10 (w/w).

In an embodiment, the pH of the mixture is adjusted to a pH of about 6.5to about 10.0. In another embodiment, the pH of the mixture is adjustedto a pH of about 7.0 to about 9.0.

In another embodiment of the disclosure, the mixture is separated bycentrifugation or hydrocyclone to separate the fiber from the mixtureand form the protein slurry. In a further embodiment, the mixture isseparated by centrifugation to separate the fiber from the mixture andform the protein slurry. In an embodiment, the mixture is centrifuged ata speed of about 1,000 rpm to about 2,000 rpm. In a further embodiment,the mixture is centrifuged at a speed of about 1,400 to about 1,600 rpm.In an embodiment, the mixture is centrifuged using a decantercentrifuge.

In another embodiment, the ratio of the fiber fraction to water is about1.0:0.5 to about 1.0:3.0 (w/w). In a further embodiment, the ratio ofthe insoluble protein fraction to water is about 1.0:1.0 to about1.0:2.0 (w/w).

In another embodiment of the disclosure, the washed fiber fraction isseparated by centrifugation or hydrocyclone to separate the fiber solidsand form second the protein slurry. In a further embodiment, the washedfiber fraction is separated by centrifugation to separate the fiber andform the second protein slurry. In an embodiment, the mixture iscentrifuged at a speed of about 1,000 rpm to about 2,000 rpm. In afurther embodiment, the fiber fraction is centrifuged at a speed ofabout 1,400 to about 1,600 rpm. In an embodiment, the fiber fraction iscentrifuged using a decanter centrifuge.

In another embodiment, the first and second slurries are filtered usingan ultrafiltration/microfiltration apparatus. In a further embodiment,the ultrafiltration/microfiltration apparatus comprises a membrane tofilter proteins larger than about 10,000 daltons. In an embodiment, theprocess further comprises the step of filtering the first and secondslurries using a diafiltration apparatus.

In another embodiment, the protein concentrate is dried in a vacuumdryer, fluidized bed dryer, ring dryer or spray dryer to form the driedprotein concentrate.

In another embodiment, the protein isolate comprises a hydrolyzedprotein isolate. In another embodiment, the protein isolate ishydrolyzed to produce peptides and free amino acids. In anotherembodiment, the hydrolyzed protein isolate comprises peptides and/orfree amino acids.

In a further embodiment, the protein concentrate comprises a proteincontent of about 30% to about 90% on a dry weight basis.

In another embodiment of the disclosure, there is also provided aprocess for the removal of fiber from a partially defatted, fullydefatted or protein-enriched algae, comprising:

-   -   i) mixing macroalgae and/or microalgae with a blending solvent,        optionally water, an aqueous solution or protein containing        solution, to form a mixture and optionally treating the mixture        with phytase at a temperature and a pH suitable for phytase        activity;    -   ii) optionally adjusting the pH of the protein slurry to a pH of        about 2 to about 10; and    -   iii) separating the mixture to form a protein slurry comprising        soluble and insoluble proteins and an insoluble fiber fraction.

The present disclosure relates to processes for the production ofprotein concentrates and protein isolates, in addition to hydrolyzedprotein concentrates and isolates, in which the macroalgae and/ormicroalgae is subjected to low g-forces to separate the fiber from theinsoluble and soluble protein fractions.

Accordingly, the present disclosure includes a process for theproduction of a protein concentrate from macroalgae and/or microalgaecomprising:

i) mixing the macroalgae and/or microalgae with a first blending solventto form a mixture;

ii) optionally treating the mixture with phytase at a temperature and apH suitable for phytase activity;

iii) optionally adjusting the pH of the mixture to a pH between 6.0 and10.0;

iv) subjecting the mixture to a g-force sufficient to separate themixture to form

-   -   a) a fiber fraction, and    -   b) protein fractions comprising an insoluble protein fraction        and a soluble protein fraction;

v) optionally mixing the fiber fraction with a second blending solventand repeating step iv);

vi) optionally adjusting the pH of the protein fractions to a pH between4.0 and 6.0;

vii) optionally heating the protein fractions to a temperature between80° C. and 100° C. to precipitate the proteins; and

viii) separating the precipitated proteins from the protein fraction toform the protein concentrate.

In another embodiment, the first and second blending solvents comprisewater, a saline solution or a polysaccharide solution. In a furtherembodiment, the first and second blending solvents comprise water.

In an embodiment of the disclosure, the ratio of the macroalgae and/ormicroalgae to the first blending solvent is 1:3 to 1:30 (w/w) of algaeto solvent, optionally about 1:8 to about 1:10 (w/w).

In an embodiment of the disclosure, the phytase is added in an amountbetween 0.01% and 0.1% (w/w) based on the weight of the macroalgaeand/or microalgae. In a further embodiment, the temperature suitable forphytase activity is between 20° and 60° C. In a further embodiment, thepH suitable for phytase activity is between 2.0 and 7.0.

In another embodiment of the disclosure, the mixture is subjected to ag-force of between 100 g and 500 g, optionally between 150 g and 400 g,suitably between 180 g and 350 g.

In another embodiment, separating the mixture comprises using acentrifuge or a hydrocyclone. In another embodiment, the centrifugecomprises a decanter centrifuge or disc stack centrifuge.

In a further embodiment, separating the precipitated proteins comprisesusing a centrifuge or a hydrocyclone. In another embodiment, separatingthe precipitated proteins comprises using a centrifuge. In anotherembodiment, centrifuging the precipitated proteins comprises a g-forcebetween 2,500 g and 9,500 g.

In another embodiment of the disclosure, the process further comprisesthe step of drying the protein concentrate to a moisture content ofbetween 4% and 8% (w/w).

In another embodiment, the protein concentrate comprises a hydrolyzedprotein concentrate. In another embodiment, the hydrolyzed proteinconcentrate comprises peptides and/or free amino acids.

The present disclosure also includes a process for the production of aprotein concentrate from macroalgae and/or microalgae comprising:

i) mixing the macroalgae and/or microalgae with a first blending solventto form a mixture;

ii) optionally treating the mixture with phytase at a temperature and apH suitable for phytase activity;

iii) optionally adjusting the pH of the mixture to a pH between 6.0 and10.0;

iv) subjecting the mixture to a g-force sufficient to separate themixture to form

-   -   a) a fiber fraction, and    -   b) protein fractions comprising an insoluble protein fraction        and a soluble protein fraction;

v) optionally mixing the fiber fraction with a second blending solventand repeating step iv);

vi) optionally adjusting the pH of the protein fractions to a pH between4.0 and 6.0;

vii) mixing the protein fractions with a mixing solvent to form aprotein slurry and precipitate the proteins;

viii) separating the precipitated proteins from the protein slurry toform the protein concentrate; and

viii) optionally repeating steps vi) and vii) with the precipitatedproteins.

In another embodiment, the first and second blending solvents comprisewater, a saline solution or a polysaccharide solution. In a furtherembodiment, the first and second blending solvents comprise water.

In an embodiment of the disclosure, the ratio of the macroalgae and/ormicroalgae to the first blending solvent is 1:3 to 1:30 (w/w) of algaeto solvent, optionally about 1:8 to about 1:10 (w/w).

In an embodiment of the disclosure, the phytase is added in an amountbetween 0.01% and 0.1% (w/w) based on the weight of the macroalgaeand/or microalgae. In a further embodiment, the temperature suitable forphytase activity is between 20° and 60° C. In a further embodiment, thepH suitable for phytase activity is between 2.0 and 7.0.

In another embodiment of the disclosure, the mixture is subjected to ag-force of between 100 g and 500 g, suitably between 150 g and 400 g,optionally between 180 g and 350 g.

In another embodiment, separating the mixture comprises using acentrifuge or a hydrocyclone. In another embodiment, the centrifugecomprises a decanter centrifuge or a disc stack centrifuge.

In another embodiment of the disclosure, the mixing solvent comprises anethanol:water mixture, wherein the ethanol is present in an amountbetween 90% and 100% (v/v).

In another embodiment, separating the precipitated proteins comprisesusing a centrifuge or a hydrocyclone. In another embodiment, separatingthe precipitated proteins comprises using a centrifuge. In anotherembodiment, centrifuging the precipitated proteins comprises a g-forcebetween 2,500 g and 9,500 g.

In another embodiment of the disclosure, steps vii) and viii) arerepeated at least twice.

In another embodiment, the process further comprises the step of dryingthe protein concentrate to a moisture content of between 4% and 8%(w/w). In another embodiment, the protein concentrate comprises ahydrolyzed protein concentrate. In a further embodiment, the hydrolyzedprotein concentrate comprises peptides and/or free amino acids.

The present disclosure also includes a process for the production of aprotein isolate from macroalgae and/or microalgae comprising:

i) mixing the macroalgae and/or microalgae with a first blending solventto form a mixture;

ii) optionally treating the mixture with phytase at a temperature and apH suitable for phytase activity;

iii) optionally adjusting the pH of the mixture to a pH between 6.0 and10.0;

iv) subjecting the mixture to a g-force sufficient to separate themixture to form

-   -   a) a fiber fraction, and    -   b) protein fractions comprising an insoluble protein fraction        and a soluble protein fraction;

v) optionally mixing the fiber fraction with a second blending solventand repeating step iv);

vi) separating the insoluble protein fraction from the soluble proteinfraction to recover therefrom an insoluble protein concentrate and asoluble protein extract; and

vii) subjecting the soluble protein extract to filtration to recovertherefrom the protein isolate.

In another embodiment, the first and second blending solvents comprisewater, a saline solution or a polysaccharide solution. In a furtherembodiment, the first and second blending solvents comprise water.

In an embodiment of the disclosure, the ratio of the macroalgae and/ormicroalgae to the first blending solvent is 1:3 to 1:30 (w/w) of algaeto water, optionally about 1:8 to about 1:10 (w/w).

In an embodiment of the disclosure, the phytase is added in an amountbetween 0.01% to 0.1% (w/w) based on the weight of the macroalgae and/ormicroalgae. In a further embodiment, the temperature suitable forphytase activity is between 20° and 60° C. In a further embodiment, thepH suitable for phytase activity is between 2.0 and 7.0.

In another embodiment of the disclosure, the mixture is subjected to ag-force of between 100 g and 500 g, suitably between 150 g and 400 g,optionally between 180 g and 350 g.

In another embodiment, separating the mixture comprises using acentrifuge or a hydrocyclone. In an embodiment, the centrifuge comprisesa decanter centrifuge or a disc stack centrifuge.

In another embodiment, separating the insoluble protein fraction fromthe soluble protein fraction comprises using a centrifuge or ahydrocyclone. In a further embodiment separating the insoluble proteinfraction from the soluble protein fraction comprises using a centrifuge.In another embodiment, centrifuging to separate the insoluble proteinfraction from the soluble protein fraction comprises a g-force between2,500 g and 9,500 g.

In another embodiment, the process further comprises the step of dryingthe protein isolate to a moisture content of between 4% and 8% (w/w).

In a further embodiment, the protein isolate comprises a hydrolyzedprotein concentrate. In another embodiment, the hydrolyzed proteinconcentrate comprises peptides and/or free amino acids.

In another embodiment of the disclosure, there is also provided aprocess for the production of a protein concentrate from macroalgaeand/or microalgae comprising:

i) mixing the macroalgae and/or microalgae with a first blending solventto form a mixture;

ii) optionally treating the mixture with phytase at a temperature and apH suitable for phytase activity;

iii) optionally adjusting the pH of the mixture to solubilize proteinsin the mixture;

iv) subjecting the mixture to a g-force sufficient to separate themixture to form

-   -   a) a fiber fraction, and    -   b) a protein fraction comprising        -   (i) an insoluble protein fraction, and        -   (ii) a soluble protein fraction;

v) separating the fiber fraction from the protein fraction and mixingthe fiber fraction with a second blending solvent to form a fibermixture;

vi) treating the fiber mixture with a protease at a temperature and a pHsuitable for protease activity;

vii) subjecting the fiber mixture to a g-force sufficient to separatethe fiber mixture to form:

-   -   a) a second fiber fraction, and    -   b) a hydrolyzed protein fraction, comprising        -   (i) an insoluble protein fraction comprising partially            hydrolyzed and un-hydrolyzed protein, and        -   (ii) a soluble hydrolyzed protein fraction;

viii) optionally adjusting the pH of the protein fraction from stepiv(b) to a pH suitable to precipitate proteins;

ix) separating the precipitated proteins from the protein fraction;

x) optionally combining the precipitated proteins and the hydrolyzedprotein fraction to form the protein concentrate.

In another embodiment, the process further comprises mixing the fiberfraction with the first blending solvent and repeating step iv) once,twice or three times and/or mixing the second fiber fraction with thesecond blending solvent and repeating step vii) once, twice or threetimes.

In another embodiment, the first and second blending solvents comprisewater, a saline solution or a polysaccharide solution, optionally water,and wherein the ratio of the macroalgae and/or microalgae to the firstblending solvent is 1:3 to 1:30 (w/w) of algae to water.

In another embodiment, the temperature suitable for phytase activity isbetween 20° and 60° C. and the pH suitable for phytase activity isbetween 2.0 and 7.0 and the temperature suitable for protease activityis between 30° and 70° C. and the pH suitable for protease activity isbetween 5.0 and 9.0.

In another embodiment, the mixture and/or the fiber mixture is subjectedto a g-force of between 100 g and 500 g, optionally between 150 g and400 g, or between 170 g and 350 g. In an embodiment, separating themixture and/or the fiber mixture comprises using a centrifuge or ahydrocyclone.

In a further embodiment, the pH suitable to precipitate the proteins inthe protein fraction is between 4.0 and 6.0.

In another embodiment, the process further comprises the step of dryingthe protein concentrate to a moisture of between 4% and 8% (w/w). Inanother embodiment, the protein concentrate also comprises peptides andfree amino acids.

The present disclosure also includes a process for the production of aprotein concentrate from macroalgae and/or microalgae comprising:

i) mixing the macroalgae and/or microalgae with a first blending solventto form a mixture;

ii) optionally treating the mixture with phytase at a temperature and apH suitable for phytase activity;

iii) optionally adjusting the pH of the mixture to solubilize proteinsin the mixture;

iv) subjecting the mixture to a g-force sufficient to separate themixture to form

-   -   a) a fiber fraction, and    -   b) a protein fraction comprising        -   (i) an insoluble protein fraction, and        -   (ii) a soluble protein fraction;

v) separating the fiber fraction from the protein fraction and mixingthe fiber fraction with a second blending solvent to form a fibermixture;

vi) treating the fiber mixture with a protease at a temperature and a pHsuitable for protease activity;

vii) subjecting the fiber mixture to a g-force sufficient to separatethe fiber mixture to form:

-   -   a) a second fiber fraction, and    -   b) a hydrolyzed protein fraction, comprising        -   (i) an insoluble protein fraction comprising partially            hydrolyzed and un-hydrolyzed protein, and        -   (ii) a soluble hydrolyzed protein fraction;

viii) mixing the protein fraction with a mixing solvent to precipitateproteins;

ix) separating the precipitated proteins from the protein fraction; and

x) optionally combining the precipitated proteins and the hydrolyzedprotein fraction to form the protein concentrate.

In another embodiment, the process further comprises mixing the fiberfraction with the first blending solvent and repeating step iv) once,twice or three times and/or mixing the second fiber fraction with thesecond blending solvent and repeating step vii) once, twice or threetimes.

In another embodiment, the first and second blending solvents comprisewater, a saline solution or a polysaccharide solution, optionally water,and wherein the ratio of the macroalgae and/or microalgae to the firstblending solvent is 1:3 to 1:30 (w/w) of algae to water.

In another embodiment, the temperature suitable for phytase activity isbetween 20° and 60° C. and the pH suitable for phytase activity isbetween 2.0 and 7.0 and the temperature suitable for protease activityis between 30° and 70° C. and the pH suitable for protease activity isbetween 5.0 and 9.0.

In another embodiment, the mixture and/or the fiber mixture is subjectedto a g-force of between 100 g and 500 g, optionally between 150 g and400 g, or between 170 g and 350 g. In an embodiment, separating themixture and/or the fiber mixture comprises using a centrifuge or ahydrocyclone.

In another embodiment, the mixing solvent comprises an ethanol:watermixture, wherein the ethanol is present in an amount between 80% and100% (v/v).

In another embodiment, the process further comprises the step of dryingthe protein concentrate to a moisture of between 4% and 8% (w/w). Inanother embodiment, the protein concentrate also comprises peptides andfree amino acids.

In another embodiment, the present disclosure also includes a processfor the production of a protein isolate from macroalgae and/ormicroalgae comprising:

i) mixing the macroalgae and/or microalgae with a first blending solventto form a mixture;

ii) optionally treating the mixture with phytase at a temperature and apH suitable for phytase activity;

iii) optionally adjusting the pH of the mixture to solubilize proteinsin the mixture;

iv) subjecting the mixture to a g-force sufficient to separate themixture to form

-   -   a) a fiber fraction, and    -   b) a protein fraction comprising        -   (i) an insoluble protein fraction, and        -   (ii) a soluble protein fraction;

vi) separating the insoluble protein fraction from the soluble proteinfraction to recover therefrom an insoluble protein concentrate and asoluble protein extract; and

vii) subjecting the soluble protein extract to membrane filtration torecover therefrom the protein isolate.

In another embodiment, the process further comprises mixing the fiberfraction with the first blending solvent and repeating step iv) once,twice or three times.

In another embodiment, the first and second blending solvents comprisewater, a saline solution or a polysaccharide solution, optionally water,and wherein the ratio of the macroalgae and/or microalgae to the firstblending solvent is 1:3 to 1:30 (w/w) of algae to water.

In another embodiment, the temperature suitable for phytase activity isbetween 20° and 60° C. and the pH suitable for phytase activity isbetween 2.0 and 7.0 and the temperature suitable for protease activityis between 30° and 70° C. and the pH suitable for protease activity isbetween 5.0 and 9.0.

In another embodiment, the mixture and/or the fiber mixture is subjectedto a g-force of between 100 g and 500 g, optionally between 150 g and400 g, or between 170 g and 350 g. In an embodiment, separating themixture and/or the fiber mixture comprises using a centrifuge or ahydrocyclone.

In another embodiment, the process further comprises the step of dryingthe protein concentrate to a moisture of between 4% and 8% (w/w). Inanother embodiment, the protein concentrate also comprises peptides andfree amino acids.

Other features and advantages of the present disclosure will becomeapparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples while indicating preferred embodiments of the disclosure aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the disclosure will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the invention will be described in relation to thedrawings in which:

FIG. 1 is a schematic representation showing a preparation of aprotein-enriched algae from a defatted algae;

FIG. 2 is a schematic representation in a first embodiment showing themilling and screening of defatted algae;

FIG. 3 is a schematic representation in a second embodiment illustratinga milling and screening process of defatted algae;

FIG. 4 is schematic representation showing a preparation of a proteinconcentrate from a protein-enriched algae;

FIG. 5 is a schematic representation in a first embodiment showing theremoval of fiber during a preparation of a protein concentrate from aprotein-enriched algae;

FIG. 6 is a schematic representation in a second embodiment showing theremoval of fiber during a preparation of a protein concentrate from aprotein-enriched algae;

FIG. 7 is a schematic representation in a third embodiment showing theremoval of fiber during a preparation of a protein concentrate from aprotein-enriched algae;

FIG. 8 is a schematic representation in a fourth embodiment showing theremoval of fiber during the preparation of a protein concentrate from aprotein-enriched algae;

FIG. 9 is a schematic representation in a first embodiment showing apreparation of a protein concentrate and a protein isolate from aprotein-enriched algae;

FIG. 10 is a schematic representation in a second embodiment showing apreparation of a protein concentrate and a protein isolate from aprotein-enriched algae;

FIG. 11 is a schematic representation in a first embodiment showing apreparation of a protein isolate from a protein-enriched algae;

FIG. 12 is a schematic representation in a second embodiment showing apreparation of a protein isolate from a protein-enriched algae;

FIG. 13 is a schematic representation in a first embodiment showing apreparation of a protein concentrate from a protein-enriched algae;

FIG. 14 is a schematic representation in a second embodiment showing apreparation of a protein concentrate from a protein-enriched algae;

FIG. 15 is a schematic representation in a third embodiment showing apreparation of a protein concentrate from a protein enriched algae;

FIG. 16 is a schematic representation illustrating a preparation of aprotein concentrate from a protein slurry with fiber removed;

FIG. 17 is a schematic representation illustrating a wet fiber removalprocess;

FIG. 18 is a schematic representation illustrating a preparation of aprotein concentrate from a protein slurry after the removal of fiber;

FIG. 19 is schematic representation illustrating a preparation of aprotein concentrate produced by recycling a protein fraction;

FIG. 20 is a schematic representation illustrating a wet fiber removalprocess;

FIG. 21 is a schematic representation illustrating a first recycling ofa protein fraction and a wet fiber removal process;

FIG. 22 is a schematic representation illustrating a second recycling ofa protein fraction and a wet fiber removal process;

FIG. 23 is a schematic representation illustrating a third recycling ofa protein fraction and a wet fiber removal process;

FIG. 24 is a schematic representation illustrating a fourth recycling ofa protein fraction and a wet fiber removal process;

FIG. 25 is a schematic representation illustrating a preparation of aprotein isolate and a hydrolyzed protein extract;

FIG. 26 is a schematic representation illustrating a preparation of ahydrolyzed protein extract;

FIG. 27 is a schematic representation illustrating a fifth recycling ofa protein fraction and a wet fiber removal process;

FIG. 28 is a schematic representation in a first embodiment illustratinga preparation of a protein concentrate;

FIG. 29 is a schematic representation in a second embodimentillustrating a preparation of a protein concentrate;

FIG. 30 is a schematic representation in a first embodiment illustratinga separation and removal of fiber from a protein slurry containinginsoluble and soluble proteins;

FIG. 31 is a schematic representation in a second embodimentillustrating a separation and removal of fiber from a protein slurrycontaining insoluble and soluble proteins;

FIG. 32 is a schematic representation in a first embodiment showing theremoval of fiber during the preparation of a protein concentrate from adefatted algae;

FIG. 33 is a schematic representation in a second embodiment showing theremoval of fiber during the preparation of a protein concentrate from adefatted algae;

FIG. 34 is a schematic representation showing the removal of fiberduring the preparation of a protein concentrate and a protein isolatefrom a defatted algae;

FIGS. 35. and 36 are schematic representations illustrating apreparation of a protein concentrate from microalage and/or macroalgaemeal using a protease;

FIG. 37 is a schematic representation illustrating a preparation of aprotein concentrate using a concurrent process; and

FIG. 38 is a schematic representation illustrating a preparation of aprotein concentrate using a counter-current process.

DETAILED DESCRIPTION OF THE DISCLOSURE (I) Definitions

The term “peptide” as used herein refers to various natural compoundscontaining two or more amino acids linked by the carboxylic acid groupof one amino acid to the amino group of another amino acid. Peptidesgenerally have 4-100 amino acids (US Patent Office Classification IndexGlossary) and a molecular weight of less than about 10,000 Daltons.

The term “protein” as used herein refers to peptides with more thanabout 50-100 amino acids and a molecular weight in excess of about10,000 Daltons. The US Patent Office Classification Index Glossarydefines protein as peptides with more then 100 amino acids.

The term “partially defatted macroalgae and/or microalgae” or “totallydefatted macroalgae and/or microalgae” as used herein refers to algaewhich has been treated to remove some, or all, of the oil containedwithin. In an embodiment, to remove oil from macroalgae and/ormicroalgae, the algae are dried. Dried macroalgae and/or microalgae aremixed with solvent, such as hexane or methyl pentane, at a ratio of 1 to3 to 1 to 10 by weight. The algae solvent slurry is milled (such as witha bead mill) to break the cell walls of the algae. This is followed bycentrifugation to separate the oil in the solvent from the solids. Thesolvent extract is desolventized and dried to produce crude algae oil.The solids are desolventized and dried to produce defatted (partially ortotally) macroalgae and/or microalgae, which can be used in theprocesses of the present disclosure. In another embodiment, wetmacroalgae and/or microalgae are wet milled, and optionally enzymatichydrolysis using cellulase, to break the cell walls. This is followed bycentrifugation to separate oil layer from aqueous layer. The oil layeris processed into oil. The aqueous layer is used as starting materialfor the processes of the present disclosure. Depending on the identityof the macroalgae and/or microalgae, the oil content may be minimal,such that it is not necessary to perform an oil extraction step.

The term “protein-enriched macroalgae and/or microalgae” as used hereinrefers to a defatted (partially or totally) macroalgae and/or microalgaeas described above, which has subsequently been treated to remove fiber.Accordingly, the macroalgae and/or microalgae is typically subjected toa milling step and a screening step to remove fiber and obtain algaehaving a higher protein content than the algae initially contained, on adry weight basis, and about 5% to about 6.5% fiber, optionally less thanabout 6%. Collectively, the terms “algae” and “macroalgae and/ormicroalgae” may be used to refer to a defatted (partially or totally)algae starting material, as well as a protein-enriched material.

The term “macroalgae and/or microalgae” (collectively known as “algae”)as used herein refers any aquatic photosynthetic organism, ranging fromsingle cell organisms, to large aquatic plants such as kelp. Microalgaerefers to (but not limited to) single-cell or groups of cells joinedtogether from the Kingdom Protista and refers to all photosyntheticprotists, such as phytoplankton, cyanobacteria, diatoms,dinoflagellates, etc. Macroalgae refers to for example, aquatic plants(having roots, stems and leaves) such as seaweed, chlorophyta,rhodophyta, phaeophya (kelps), etc. Different species of macroalgaeand/or microalgae contain different amounts of protein initially intheir cell structure, optionally greater than 10% (w/w), or 15% (w/w),or 20% (w/w), 40% (w/w), 50% (w/w) or 60% (w/w) or more. The macroalgaeand/or microalgae that is useful to produce a protein concentrate orisolate using the processes of the present disclosure, are those speciesin which a protein concentrate having at least 30% (w/w) is produced.

The term “protein concentrate” as used herein refers to macroalgaeand/or microalgae that has been treated to isolate protein using theprocesses of the present disclosure to increase the protein content,where the protein concentrate has greater than 30% protein content,optionally greater than 50%, optionally greater than 60% but less than90% protein content on a dry weight basis. The balance may comprisecarbohydrate, ash, fiber and oil. In an embodiment, the proteinconcentrate is generally produced from the insoluble protein fraction orsoluble/insoluble protein fractions of a protein mixture. In oneembodiment, the protein concentrate also includes hydrolyzed proteinconcentrate.

The term “hydrolyzed protein concentrate” as used herein refers to aprotein concentrate that has been treated to hydrolyze the proteinswithin the protein concentrate into amino acids and smaller peptides.Proteins can be hydrolyzed using various chemicals, such as strong acidsand bases, and enzymes, preferably proteases.

The term “protein isolate” as used herein refers to macroalgae and/ormicroalgae that has been treated to isolate protein using the processesof the present disclosure to increase the protein content, where theprotein isolate has 90% or greater than 90% protein on a dry weightbasis. The balance may comprise carbohydrate, ash, and oil. In anembodiment, the protein isolate is generally produced from the solubleprotein fraction of a protein mixture.

The term “hydrolyzed protein isolate” as used herein refers to a proteinisolate that has been treated with proteases to hydrolyze the proteinswithin the protein isolate into amino acids and smaller peptides.

The term “protease” as used herein refers to any enzyme that hydrolyzesproteins by hydrolysis of the peptide bonds that link amino acidstogether in the polypeptide chain forming the protein. Examples ofproteases include, but are not limited to, Alcalase®, Flavourzyme® andProtamex®. The proteases solubilize and partially hydrolyse proteinstrapped within the fiber fraction and to release insoluble proteins fromthe fiber fraction using a dosage of a single protease.

The term “mixing solvent” as used herein refers to a solvent that formsa protein slurry or mixture when mixed with macroalgae and/ormicroalgae. In addition, the fiber present in the algae possessesminimal solubility in the mixing solvent (eg. typically less than 1%(w/w) solubility, or about 0% solubility), and suitably, is not solublein the mixing solvent. Examples of mixing solvents include, but are notlimited to, water, alcohols, such as methanol, ethanol or isopropanol,polysaccharide solutions such as guar gum solution, saline solutions, ormixtures of any of the above.

The term “blending solvent” as used herein refers to any aqueous solvent(typically at least: 80%, 85%, 90%, 95%, 98% or 99% water by weight)that forms a slurry or mixture when mixed with macroalgae and/ormicroalgae. Typically the blending solvent is free from organicsolvents, such as methanol, ethanol, propanol, iso-propanol,tetrahydrofuran since these solvents are not desirable as residues in aprotein isolate, concentrate or hydrosylate for human consumption,however, if organic solvents are present, they are in the blendingsolvent in small amount (eg. typically equal to or less than: 20%, 10%,10%, 5% or 1%) so that their presence in the final product isnegligible. Examples of blending solvents include water, acidic water,alkaline water, saline salt solutions (such as sodium chloride,potassium chloride, calcium chloride), polysaccharide solutions (such asguar gum), and aqueous protein solutions.

The disclosure contemplates using a variety of solvents, which couldinclude blending solvents, mixing solvents or other combinations oralcohols (eg. 80% ethanol), water and/or aqueous solvents. The use ofthe term blending solvents should not be construed as precluding the useof organic solvents in processes as disclosed herein.

The term “extraction solvent” as used herein refers to a solvent whichis capable of solubilizing antinutritional compounds, or otherconstituents, that are present in the algae and which are desirablyremoved. Examples of antinutritionals include, but are not limited to,glucosinolates, phytic acid, phytates and other compounds that reducethe nutritional or commercial value of the protein concentrate orprotein isolate. Antinutritional compounds are compounds that are, forexample, not digestible by mammals (e.g humans), have adverse effects,such as toxicity or are bitter tasting, and are desirably removed fromthe protein product. Accordingly, the concentration of antinutritionalsin a protein product produced in accordance with a process of thepresent disclosure is less than about 1% (w/w), optionally less thanabout 0.5% (w/w), optionally less than about 0.1% (w/w), and optionallyless than about 0.05% (w/w). Examples of other compounds include, butare not limited to, materials that undesirably effect the quality,color, taste, odor, appearance or characteristics of the end product.Examples include compounds that cause a darkening or variation in thecolor, cause a bitter taste or a pungent odor, such as sinapine orsinigrin, or affect the handling or agglomeration of the end product.While the antinutritionals or other components are not desirable in theprotein concentrates or isolates they may constitute commerciallyvaluable side products which can have utility as medicinal or industrialingredients or end products once separated from the protein concentrateor isolate. Examples of extraction solvents include, but are not limitedto, water, alcohols, such as methanol, ethanol, isopropanol, or mixturesof any of the above. Other extractions solvents which are useful includetetrahydrofuran (THF), dimethylformamide (DMF), and ethers, such asmethyl t-butyl ether. However, it will be known to those skilled in theart that solvents such as THF, DMF or ethers, as a result of theirhigher toxicity as compared to, for example, ethanol, require lowerlimits in the protein product. macroalgae and/or microalgae

The term “homogeneous agitation” as used herein refers to the mixing ofmacroalgae and/or microalgae with a solvent to form a homogenous mixtureor suspension. Such agitation is accomplished, for example, by mixingthe slurry or mixture at a speed of about 30 rpm to about 300 rpm in astandard mixer.

The term “washed” used herein refers to a protein fraction that has beenmixed with an extraction solvent, such as ethanol, to removeantinutritional compounds, or other constituents, from the proteinfraction.

The term “protein slurry” as used herein refers to protein, for example,the protein in macroalgae and/or microalgae, that has been mixed with amixing solvent to form a suspension of protein, and optionally fiber andother antinutritional compounds, in the mixing solvent.

The terms “soluble protein fraction” and “insoluble protein fraction” asused herein refer to specific protein fractions which are either solubleor insoluble, respectively, in a particular solvent, such as a blendingsolvent, mixing solvent or an extraction solvent. In an embodiment, theinsoluble protein fraction is generally composed of insoluble globulinand denatured proteins. The insoluble protein fraction is generallycomposed of insoluble globulin proteins. In another embodiment, thesoluble protein fraction is generally composed of albumin, solubleglobulin and undenatured proteins. The soluble protein fraction isgenerally composed of soluble albumin and soluble globulin proteins.

The term “water” as used herein refers to any source of water, forexample, tap water, distilled water or reverse osmosis water.

The term “alkaline water” as used herein refers to water which has abasic pH of greater than about 7.0, optionally about 7.0 to about 12.0.The alkalinity of the water results from the addition of a base towater, for example, an alkali hydroxide such as sodium hydroxide. Forexample, a solution of sodium hydroxide at a concentration of about 5%to about 15% (w/w), optionally 11%.

The term “suitable for phytase activity” as used herein refers to theconditions, such as the temperature and pH, and optionally includes thelength of time, in which the phytase enzyme is able to hydrolyze thephosphate groups on phytate or phytic acid, and accordingly, reduce theamount of phytates or phytic acid in the mixture. In an embodiment, thetemperature suitable for phytase activity is between 20° C. and 60° C.,optionally between 40° C. and 55° C., suitably between 50° C. and 55° C.In another embodiment, the pH suitable for phytase activity is between2.0 and 7.0, optionally between 4.0 and 6.0, suitably between 4.5 and5.5, optionally 5.0 to 5.5. In another embodiment, the concentration ofthe phytase enzyme is between 0.01% to 1.0% (w/w) based on the weight ofthe macroalgae and/or microalgae, optionally 0.01% and 0.5% optionally0.01% and 0.1%. It will be understood that the conditions suitable forphytase activity apply to all of the processes of the presentdisclosure.

The term “suitable for protease activity” as used herein refers to theconditions, such as the temperature and pH, and optionally includes thelength of time, in which a protease enzyme is able to hydrolyzeproteins. In an embodiment, the temperature suitable for proteaseactivity is between 30° C. and 70° C., optionally between 35° C. and 70°C. In another embodiment, the pH suitable for protease activity isbetween 5.0 and 9.0, optionally between 5.5 and 8.5. In anotherembodiment, the concentration of the protease enzyme is between 0.01% to1.0% (w/w) based on the weight of the macroalgae and/or microalgae,optionally 0.01% and 0.5% optionally 0.01% and 0.1%. It will beunderstood that the conditions suitable for protease activity apply toall of the processes of the present disclosure.

The term “g-force sufficient to separate the mixture” as used hereinrefers to the force necessary to separate the insoluble fiber fractionin the mixture from the protein fractions. In an embodiment, the g-forceis between 100 g and 500 g, suitably between 150 g and 400 g, optionallybetween 170 g and 350 g. It will be understood that when the mixture issubjected to a sufficient g-force, the insoluble fiber, due to itsrelative higher density and/or greater particle size, will separate fromthe protein fractions. It should also be recognized that forces greaterthan the ranges necessary to separate the phases are not desirable asthey can result in the high concentrations of the insoluble proteinbeing deposited in the fiber phase. In addition, as a result of theinsoluble protein fraction having a higher relative density and/orparticle size compared to the soluble protein, the insoluble proteinfraction will separate from the soluble protein fraction. It will beunderstood though that not all of the protein will separate from theinsoluble fiber fraction, and likewise, not all of the insoluble fiberwill separate from the protein fraction. Moreover, not all of theinsoluble protein fraction will separate from the soluble proteinfraction. Accordingly, when the mixture has been subjected to a g-forcesufficient to separate the mixture, the insoluble fiber fraction willcomprise at least 10% crude fiber, optionally 15%, 20%, 25, 30% crudefiber on a dry weight basis. Likewise, the protein fraction willcomprise less than 10% crude fiber, optionally less than 5%, 4%, 3%, 2%,1% and less than 1% crude fiber with the majority of other materialcomprising soluble and insoluble proteins, carbohydrate, ash and oil. Inan embodiment, the g-force sufficient to separate the mixture isobtained by rotating a centrifuge at a speed of about 500 RPM to about2,500 RPM. It will be understood that a centrifuge will have arotational radius which will vary depending on the size of thecentrifuge. In another embodiment, the g-force sufficient to separatethe mixture is obtained by using a hydrocyclone with a g-force ofbetween 50 g and 250 g.

(II) Protein Concentrates and Isolates

The present disclosure relates to processes for the production of aprotein concentrate or a protein isolate from macroalgae and/ormicroalgae. A protein concentrate is an isolated protein extract ofmacroalgae and/or microalgae, wherein the extract has greater than 30%protein content but less than 90% protein content on a dry weight basis.A protein concentrate has been treated to separate protein in themacroalgae and/or microalgae from the fiber and other unwantedantinutritional factors. A protein isolate is an isolated proteinextract of macroalgae and/or microalgae, wherein the extract has greaterthan or equal to 90% protein content on a dry weight basis. Typically,the protein isolate has up to 98%, 99%, 99.5% or 100% protein content ona dry weight basis. Typically, the non-protein content includesnon-protein compounds such as antinutritional substances, fiber, andother components or impurities such as coloring agents.

In an embodiment, the disclosure provides a process for the removal offiber, antinutritionals and other constituents, that are present withinthe algae. A person skilled in the art would recognize thatantinutritionals include glucosinolates, phytic acid, phytates and othercompounds that reduce the nutritional or commercial value of the proteinconcentrate or protein isolate. For example, antinutritional compoundsmay not be digestible by mammals (e.g humans), have adverse effects,such as toxicity, and are desirably removed from the protein product.Certain antinutritionals have other undesirable properties, such asundesirable organoleptic properties. Examples of such compounds aresinapine, which has a bitter taste, and sinigrin which has a pungent andvery bitter flavor. Further, other antinutritional constituent of algaethat are typically removed include, but are not limited to, coloringagents and/or other inert compounds. In an embodiment, the constituentswhich are removed or are reduced to safe or acceptable levels, areundesirable constituents or impurities using the processes of thepresent disclosure. A person skilled the art would recognize the safeand/or acceptable levels of particular antinutritionals in the finalprotein product.

In another embodiment of the disclosure, there is also included proteinconcentrates and protein isolates, produced in accordance with theprocesses of the disclosure.

In an embodiment, the protein concentrate possessing a protein contentof about 30% to about 70% produced in accordance with the processes ofthe present disclosure are utilized as a protein ingredient in aquafeedsfor fish, swine and pet foods.

In another embodiment, the protein concentrate possessing a proteincontent of about 30% to about 75% produced in accordance with theprocesses of the present disclosure are useful as a protein ingredientfor baked food products such as bread, rolls, cake and pastry products(including mixtures for preparing baked food products), cookies,biscuits, crackers, pancakes, pastries, doughnuts, and other pastaproducts. In addition, this protein concentrate is useful as a proteiningredient in meat products such as baked meat, hot dogs, bologna,analogs, ham and sausages. Further, this protein concentrate is alsouseful as a protein ingredient in vegetarian foods. It will beunderstood by a person skilled in the art that this protein concentrateis also useful for other applications where a lower grade of proteinconcentrate is sufficient, such as in aquafeeds and pet foods asdescribed above.

In another embodiment, the protein concentrate possessing a proteincontent of about 30% to less than 90% produced in accordance with theprocesses of the present disclosure is useful as a protein ingredient inbreakfast cereals, and baked goods, as well as meat products such asbologna, frankfurters, luncheon loaves and ham. Further, this proteinconcentrate is useful in candies, confections, desserts, dietary items,Asian foods, soup mixes, gravies and other similar food items. Again, itwill be understood by a person skilled in the art that this proteinconcentrate is also useful for other applications where a lower grade ofprotein concentrate is sufficient, such as in aquafeeds, pet foods,bakery products and meat products, as described above.

In another embodiment, the protein isolate possessing a protein contentof greater than 90% produced in accordance with the processes of thepresent disclosure is useful as a protein ingredient in nutritionalbeverages such as protein fortified soft drinks, sports drinks, fruitjuices and other high protein drinks. In addition, this protein isolateis useful as a protein ingredient for nutritional supplements, specialdiet products, and high protein nutritional tablets. In addition, theprotein isolate is useful as a protein ingredient in infant formulas, aswell as an ingredient in comminuted and emulsified meats, simulatedmeats, combination meat products and cured or uncured meat products.Further, the protein isolate is useful as a protein ingredient in pasta(eg. macaroni), bread and other bakery products, pancakes, waffles,crackers, donuts, pie crusts, soups, egg replacements, dried milkreplacements and dairy analogs. Again, it will be understood by a personskilled in the art that this protein isolate is also useful for otherapplications where a lower grade of protein is sufficient, such as inaquafeeds, pet foods, and meat products, as described above.

In another embodiment, the hydrolyzed protein isolate possessing aprotein content of greater than 90% produced in accordance with theprocesses of the present disclosure is useful as a protein ingredient innutritional beverages such as protein fortified soft drinks, sportsdrinks, fruit juices and other high protein drinks. In addition, thehydrolyzed protein isolate is useful as a cosmetic ingredient. Further,the hydrolyzed protein isolate is useful as a protein ingredient forhealthy food applications to improve absorption and digestibility.Again, it will be understood by a person skilled in the art that thishydrolyzed protein isolate is also useful for other applications where alower grade of protein is sufficient, such as in aquafeeds, pet foods,bakery products and meat products, as described above.

(III) Processes of the Disclosure

A person skilled in the art would understand that depending on theidentity of the macroalgae and/or microalgae, an oil extraction step mayor may not be required. When an oil extraction is required, the algaeare dried. Dried macroalgae and/or microalgae are mixed with solvent,such as hexane or methyl pentane, at a ratio of 1 to 3 to 1 to 10 byweight. The algae solvent slurry is milled (such as with a bead mill) tobreak the cell walls of the algae. This is followed by centrifugation toseparate the oil in the solvent from the solids. The solvent extract isdesolventized and dried to produce crude algae oil. The solids aredesolventized and dried to produce defatted (partially or totally)macroalgae and/or microalgae, which are used in the processes of thepresent disclosure. In another embodiment, wet macroalgae and/ormicroalgae are wet milled, and optionally enzymatic hydrolysis usingcellulase, to break the cell walls. This is followed by centrifugationto separate oil layer from aqueous layer. The oil layer is processedinto oil. The aqueous layer is used as starting material for theprocesses of the present disclosure. Depending on the identity of themacroalgae and/or microalgae, the oil content may be minimal, such thatit is not necessary to perform an oil extraction step.

A general method for obtaining a protein-enriched algae is shown inFIGS. 1-3. For example, when beginning with alage, the moisture contentof the algae is adjusted. The moisture adjusted algae is optionallyexposed to a heat treatment. In an embodiment of the processes of thepresent disclosure, the algae is heat treated to a temperature of about60° C. to about 120° C., optionally about 70° C. to about 100° C., orabout 80° C. to about 90° C., or about 80° C. In another embodiment, theheat treatment is carried out at a temperature of 100° C. The heattreatment of the algae results in the inactivation of the enzymespresent in the algae, for example, myrosinase, lipase, phospholipase. Ifthe algae is not heat treated, the enzymes (such as myrosinase, lipase,phospholipase), as a result of their enzymatic action, can degrade theoil and breakdown glucosinolates releasing sulphur into oil. At atemperature of about 75-100° C., the enzymes are deactivated, and aretherefore not able to degrade the oil and breakdown glucosinolatesreleasing sulphur into oil. Accordingly, in an embodiment, a heattreatment temperature of 75-100° C. results in a reasonably high proteindispersibility index (PDI), lower sulphur, FFA and phosphorus in pressedand butane/R134a extracted oils.

Alternatively, in an embodiment, the algae is not exposed to a heattreatment and its moisture content is not adjusted. It will beunderstood by a person skilled in the art that the moisture content ofthe algae is typically in the range of about 7% to about 10% for apressing operation. If the moisture content of the algae is not in thisrange, the moisture of the algae is optionally adjusted to about 7% toabout 10% by adding water or drying, which is followed by blending andtempering.

The algae is then subjected to a milling step and a screening step toobtain a protein-enriched algae.

The algae is typically milled, for example with a disc mill or a hammermill, to reduce the particle size of the algae. When using a disc mill,the algae is forced through two rotating discs which crush the algae.When a hammer mill is used to reduce the particle size of the algae, thealgae is loaded into the hammer mill, wherein the hammers reduce theparticle size of the algae.

After the particle size of the algae has been sufficiently reduced, themilled algae is screened through mesh screens, which results in aninitial separation of a fiber fraction from the algae, resulting in aprotein-enriched algae. Fiber tends to have a larger particle size whichis not able to pass through the screen. However, a portion of the fiberwill be able to pass through the screen, and as such, only a portion ofthe fiber is removed by screening. Typically, about a 45 US mesh screenis used for the initial fiber separation. This is a dry screeningprocess which results in a fiber enriched algae, which does not passthrough the screen, and the protein-enriched algae, which does passthrough the screen. The protein-enriched algae, however, still containsa significant amount of fiber and other antinutritional factors. In anembodiment of the disclosure, it is this protein-enriched algae that isutilized to produce the protein concentrates and protein isolates of thepresent disclosure. However, in another embodiment, it will be apparentto those skilled in the art that macroalgae and/or microalgae, apartially or totally defatted algae or a protein-enrcihed algae isutilized with the processes of the present disclosure. The use of suchmacroalgae and/or microalgae, and processing with a minimum amount ofheat during conditioning, pressing, solvent extraction, desolventizationand drying, leads to better protein concentrates and protein isolates.

In an embodiment of the present disclosure, there is a process forremoving fiber from macroalgae and/or microalgae. In particular, theprocess relates to separating and removing fiber from algae based on thedensity and particle size differences between the fiber particles andthe protein particles. The separation and removal of fiber isaccomplished by using separation methods, at specific speeds, which canseparate particles based on their density or particle size such ascentrifugation, gravity sedimentation, a gravity table or hydrocycloneto separate the fiber from the mixture and form the protein slurry. Inan embodiment, the separation is accomplished using centrifugation. Inanother embodiment, the separation is accomplished using a decantercentrifuge. In another embodiment, the separation is accomplished usinga decanter centrifuge at a speed of about 1,000 rpm to about 2,000 rpm.In another embodiment, the separation is accomplished using a decantercentrifuge at a speed of about 1,500 rpm. In an embodiment, thecentrifugation of a algae mixture results in three layers: i) aninsoluble fiber layer and a protein slurry on top of the fiber, which iscomprised of ii) an insoluble protein fraction and iii) a solubleprotein fraction. Separation of the top and middle layers (the solubleprotein extract and the insoluble fine protein fraction) from the bottomlayer (coarse fiber solids), results in a protein slurry with fiberremoved.

In an embodiment of the present disclosure, a process for the productionof a protein concentrate possessing a protein content of about 30% toabout 60% is obtained from macroalgae and/or microalgae. An optionalgeneral process for the production of a protein concentrate isillustrated in FIG. 4.

In an embodiment, macroalgae and/or microalgae is produced by theprocess above, and is then washed at least once with about 5% to about100%, optionally about 20% to about 90%, or about 40% to about 80% (v/v)ethanol in water, resulting in an ethanol extract and an ethanol washedmacroalgae and/or microalgae. Other alcohols, such as methanol orisopropanol, can be utilized for washing the macroalgae and/ormicroalgae. In an embodiment, ethanol is used for washing the macroalgaeand/or microalgae because it is less toxic than other alcohols, and ahigher percentage of ethanol residue is allowed in the final product.

In another embodiment, the macroalgae and/or microalgae is washed oncewith ethanol, wherein the ratio of ethanol to the protein-enriched algaeis about 1:3 to about 1:15, typically about 1:4 to about 1:8, optionally1:6, on a weight-to-weight basis of protein-enriched algae to ethanol.

In another embodiment, the macroalgae and/or microalgae is washed twicewith ethanol, wherein the amount of ethanol added to theprotein-enriched algae results in a ratio of about 1:2 to about 1:15,typically about 1:5 to about 1:8, optionally 1:6, on a weight-to-weightbasis of protein-enriched algae to ethanol. Typically, washing themacroalgae and/or microalgae at least twice results in the removal ofmore impurities from the macroalgae and/or microalgae and thereforeincreases the protein content in the protein concentrate.

In a further embodiment, the macroalgae and/or microalgae is washed in acounter-current extractor. In this embodiment, the macroalgae and/ormicroalgae is washed about 2 times to about 10 times, wherein the ratioof solvent to the macroalgae and/or microalgae is about 1 to about 10 ofalgae to about 1 of algae.

In another embodiment, the macroalgae and/or microalgae is washed withethanol at a temperature of about 10° C. to about 90° C., optionally 20°C. to about 60° C., suitably at a temperature of about 40° C. to about60° C.

The ethanol extract is optionally separated from the ethanol washedmacroalgae and/or microalgae by centrifugation, filtration, vacuumfiltration, pressure filtration, sedimentation, decantation or gravitydraining. With respect to centrifugation, the ethanol mixture istypically fed to a decanter centrifuge or a basket centrifuge. Theethanol extract is then separated from the ethanol washed macroalgaeand/or microalgae by centrifugal force. For the decanter centrifuge, ascrew conveyer is contained within a solid bowl and both rotate at highspeeds. Solids settling on the bowl are conveyed by the screw conveyerout of the centrifuge. For a basket centrifuge, which consists of aperforated basket rotating inside a stationary housing, the ethanolmixture is fed into the basket and centrifugal force pushes it againstthe filter liner. The solids are retained by the liner while the liquidpasses through. For filtration, the ethanol extract is typicallyseparated from the ethanol washed macroalgae and/or microalgae bydraining through a perforated belt or basket in a reactor. For vacuumfiltering or pressure filtering, the separation is aided by vacuum orpressure. In an embodiment, the ethanol extract is concentrated byevaporation of the ethanol to form a high sugar fraction, optionallycontaining antinutritional factors that can be further purified. Theantinutritional compounds may be purified into valuable pharmaceutical,medicinal or chemical compounds, such as glucosinolates, phytic acid orphytates, sinapine and sinigrin. In an embodiment, the ethanol extractis heated under vacuum at about 30° C. to about 90° C., which results inthe evaporation of ethanol and water, and soluble solids are leftbehind. Ethanol is further separated from water by distillation andre-used in the process. The concentrated high-sugar fraction is dried byspray drying, rotary drum drying, vacuum drying, flash drying, ringdrying, microwave drying, freeze drying or using a fluidized bed dryer.

In another embodiment, the washed macroalgae and/or microalgae is driedto form the protein concentrate, possessing a protein content of about30% to about 70%. In a further embodiment, the washed protein-enrichedalgae is dried in a spray dryer, drum dryer, vacuum dryer, fluidized beddryer or ring dryer to form the protein concentrate possessing a proteincontent of about 30% to about 70%. These dryers remove the solvent bydrying the protein concentrate under a vacuum or at atmospheric pressureat elevated temperatures of about 30° C. to about 100° C.

In an embodiment, the protein concentrate is dried to a moisture contentof about 1% to about 10%, optionally about 4% to about 8%.

In another embodiment, the ethanol that is removed through drying isrecovered and recycled so it can be used again in further ethanolextractions. The ethanol is recovered through evaporation anddistillation.

In another embodiment, the dried protein concentrate possessing aprotein content of about 30% to about 70% is further milled into powderform without coarse particles.

In another embodiment of the present disclosure, there is provided aprocess for producing a protein concentrate possessing a protein contentof about 30% to about 75% on a dry weight basis. In an embodiment, ageneral process for the production of a protein concentrate possessing aprotein content of about 30% to about 75% is illustrated in FIGS. 5-8,where the removal of fiber is also detailed. In an embodiment, the useof an extraction solvent, such as ethanol, leads to a proteinconcentrate or protein isolate having superior organoleptic properties,as well as superior water solubility properties, which thereforepossesses better functional properties.

Accordingly, in an embodiment of the present disclosure, a process forthe production of a protein concentrate from macroalgae and/ormicroalgae is disclosed, comprising:

-   -   1) removing fiber from the defatted or protein-enriched        macroalgae and/or microalgae, comprising either:        -   i) mixing the macroalgae and/or microalgae with a mixing            solvent to form a first mixture;            -   screening the first mixture through a mesh screen of                about 10 to about 200 US mesh size to remove the fiber;                or        -   ii) mixing the macroalgae and/or microalgae with water to            form a second mixture;            -   optionally adjusting the pH of the second mixture to a                pH of about 3 to about 7; and            -   adding cellulase complex to the second mixture and                heating to a temperature of about 30° C. to about 60° C.                to hydrolyze the fiber;    -   2) washing the first or second mixture with an extraction        solvent to form an extract and a washed macroalgae and/or        microalgae;    -   3) separating the extract from the washed defatted or        protein-enriched algae;    -   4) optionally repeating steps 2) and 3) at least one more time;        and    -   5) desolventizing the washed macroalgae and/or microalgae to        form a protein concentrate.

In an embodiment of the present disclosure, the mixing solvent is anysolvent which forms a slurry with the macroalgae and/or microalgae whenmixed together and is able to suspend the protein within the mixture. Inanother embodiment, the mixing solvent comprises water, methanol,ethanol, or isopropanol, or mixtures thereof. In a further embodiment,the mixing solvent comprises water or ethanol, and mixtures thereof.

In an embodiment of the disclosure, the macroalgae and/or microalgae ismixed with a mixing solvent in a ratio of about 3 to about 10 partssolvent to about 1 part of the macroalgae and/or microalgae, on aweight-to-weight basis.

In an embodiment of the present disclosure, the pH of the first mixtureis adjusted to a pH of about 3.0 to about 10.0, optionally about 6.8 toabout 7.2 with a solution of an alkali metal base or an acid, such asphosphoric, hydrochloric or sulphuric acid. In a further embodiment, asolution of an alkali metal base comprising about 1% to about 40% byweight, optionally about 5% to about 30%, of the alkali metal base andwater is added to the first mixture. In another embodiment, the alkalimetal base comprises sodium hydroxide (NaOH).

In another embodiment of the present disclosure, the first mixture isthoroughly agitated. In another embodiment, an inline mixer is used forthorough mixing of the first mixture. Thorough mixing of the firstmixture disperses the protein particles and releases natural sugarcompounds that are trapped inside the insoluble protein particles in themixing solvent. In addition, the agitation suspends the solids of theprotein-enriched algae in the mixing solvent.

In a further embodiment, the thoroughly mixed first mixture is wetscreened resulting in a separation of the fiber from the mixture whichcontains the protein. In another embodiment of the disclosure, the meshscreen comprises a US mesh screen of about 20 to about 200 US mesh. In afurther embodiment, the mesh screen is a vibratory screen. A personskilled in the art would recognize that other screens, for examplerevolving screens, shaking screens or oscillating screens, could be usedin place of vibratory screens to perform substantially the same functionof vibrating the mixture which aids in separation of the first mixturefrom the fiber. In an embodiment of the disclosure, the fiber in thealgae swells upon addition of the mixing solvent, increasing theparticle size of the fiber. Consequently, the mesh screen prevents thefiber from passing through, while the protein in the first mixturepasses through the screen, resulting in a separation of the fiber fromthe protein. In an embodiment, the fiber fraction is dried and can beused in dietary fiber products. The fiber fraction optionally containsprotein and carbohydrates.

In another embodiment of the present disclosure, the macroalgae and/ormicroalgae is thoroughly mixed with water to form the second mixture. Inan embodiment, wet milling is used to mix the second mixture. In anotherembodiment, an inline mixer is used to thoroughly mix the secondmixture. In an embodiment, the mixing of the macroalgae and/ormicroalgae in water, results in the internal fiber structure beingexposed, which allows for the cellulase complex to efficiently hydrolyzethe fiber.

In a further embodiment of the disclosure, the pH of the second mixtureis optionally adjusted with an acid. In an embodiment, the pH of thesecond mixture is adjusted to a pH that is suitable for the activity ofan enzyme within the second mixture. In an embodiment, the pH of thesecond mixture is adjusted to a pH of about 3 to about 7. The pH of thesecond solution is adjusted with an acid solution. In an embodiment, theacid solution is phosphoric acid, hydrochloric acid or sulfuric acid. Inan embodiment, the natural pH of the second mixture is about 6.8 toabout 7.2, and therefore the pH of the second mixture is not adjusted.

In another embodiment of the present disclosure, the cellulase complexis added to the second mixture in an amount of about 1 to about 10 grams(about 0.1% to about 1%) to about 1 kg of dried solids in the secondmixture. In a further disclosure, the cellulase complex is mixed withthe second mixture for about 0.5 hours to about 5 hours. In anotherembodiment, the cellulase complex is mixed with the second mixture forabout 1 to about 3 hours. It will be apparent to those skilled in theart that cellulase complex contains different types of cellulase enzyme.For example, cellulase complex contains at least one of endocellulase,exocellulase, cellobiohydrolase, cellobiase, endohemicellulase andexohemicellulase. Cellulase enzymes possess enzymatic activity which areable to hydrolyze the fiber to constituent sugars within the secondmixture.

In another embodiment of the present disclosure, the first or secondmixture is washed at least once with about 5% to about 100%, optionallyabout 25% to about 85%, or about 50% to about 85%, or about 60% to about85%, of the extraction solvent (v/v) in water. The addition of theextraction solvent precipitates proteins in the first or second mixture,while the carbohydrates from the algae from the hydrolyzation of thefiber remain in the extraction solvent, which allows for separation. Itwill be understood that an extraction solvent will be any solvent whichdissolves the carbohydrates and other undesirable compounds, butprecipitates the protein. In embodiment, the extraction solvent iswater, methanol, ethanol or isopropanol, and mixtures thereof. Inanother embodiment, the extraction solvent is ethanol. It will beunderstood by a person skilled in the art that if the extraction solventcomprises 100% extraction solvent, no water will be present in theextraction solvent. For example, the extraction solvent could be 100%ethanol. In another embodiment, the extraction solvent is 60% ethanol inwater.

In an embodiment of the present disclosure, the extraction solvent isadded in an amount to adjust the ratio of the extraction solvent to thefirst or second mixture of about 5% to about 95%, optionally about 10%to about 90%, or about 40% to about 80% (v/v) of the extraction solvent.

In an embodiment of the present disclosure, the first or second mixtureis washed with an extraction solvent at a temperature of about 10° C. toabout 90° C. In another embodiment, the first or second mixture iswashed with the extraction solvent at a temperature of about 20° C. toabout 60° C. In a further embodiment, the first or second mixture iswashed with the extraction solvent at a temperature of about 20° C. toabout 25° C.

In another embodiment of the present disclosure, the extract isseparated from the washed macroalgae and/or microalgae bycentrifugation, vacuum filtration, pressure filtration, decantation orgravity draining. In an embodiment, the extract is concentrated byevaporation of the extraction solvent dried to form a high sugarfraction, as is performed above.

In another embodiment of the disclosure, steps 2) and 3) are optionallyrepeated at least once. In an embodiment, steps 2) and 3) are repeatedat least twice. Repeating steps 2) and 3) results in a protein productcontaining less impurities, such as fiber and other antinutritionalfactors.

In another embodiment, the washed macroalgae and/or microalgae is driedto form the protein concentrate, possessing a protein content of about50% to about 75% on a dry weight basis. In a further embodiment, thewashed macroalgae and/or microalgae is dried in a vacuum dryer,fluidized bed dryer, spray dryer or ring dryer to form the proteinconcentrate possessing a protein content of about 30% to about 75%.

In another embodiment, the washed macroalgae and/or microalgae is driedto a moisture content of about 0.5% to about 12%, optionally about 1% toabout 10%, or about 4% to about 8%. In a further embodiment, the washedmacroalgae and/or microalgae is dried to a moisture content of about 6%.

In another embodiment of the disclosure, the extraction solvent that isremoved through drying is recovered and recycled so it can be used againin further extractions.

In another embodiment, the dried protein concentrate possessing aprotein content of about 30% to about 75% is further milled into powderform.

In another embodiment of the present disclosure, there is disclosed aprocess for the production of a protein concentrate comprising a proteincontent of about 30% to less than 90% on a dry weight basis. In anembodiment, a general process for the production of a proteinconcentrate possessing a protein content of about 30% and a proteinisolate having a protein content greater than 90% is illustrated inFIGS. 9-10.

Accordingly, a process for the production of a protein concentrate frommacroalgae and/or microalgae is disclosed, comprising:

removing fiber from the defatted or protein-enriched macroalgae and/ormicroalgae, comprising:

-   -   i) mixing the macroalgae and/or microalgae with a mixing solvent        to form a mixture;        -   optionally screening the mixture through a mesh screen of            about 10 to about 200 US mesh size to remove fiber,        -   optionally adjusting the pH of the mixture to a pH of about            7;        -   optionally milling the mixture;        -   centrifuging the mixture to remove fiber,        -   and forming a protein slurry; and    -   ii) centrifuging the protein slurry to form a protein        precipitate and a soluble protein fraction;    -   iii) washing the protein precipitate with an extraction solvent        at least once and centrifuging to form a purified protein        precipitate;    -   iv) drying the purified protein precipitate to form the protein        concentrate.

It will be understood by a person skilled in the art that the steps ofthe process do not have to be followed exactly. For example, a personskilled in the art would recognize that the milling step could beperformed before the screening step.

In an embodiment of the disclosure, the mixing solvent is any solventwhich forms a slurry with the macroalgae and/or microalgae and is ableto suspend the protein within the mixture. In another embodiment, themixing solvent comprises water, methanol, ethanol, or isopropanol, andmixtures thereof. In a further embodiment, the solvent comprises wateror ethanol, and mixtures thereof.

In an embodiment, the macroalgae and/or microalgae is mixed with themixing solvent to form a mixture in a ratio of macroalgae and/ormicroalgae to mixing solvent of about 1:3 to about 1:20, optionallyabout 1:6 to about 1:10, or about 1:6 to about 1:8.

In a further embodiment, the mixture is wet screened resulting in aseparation of the fiber from the mixture which contains the protein. Inanother embodiment of the disclosure, the mesh screen comprises a USscreen of size about 20 to about 200 mesh. In a further embodiment, themesh size is 40 US mesh size. In a further embodiment, the mesh screenis a vibratory screen. The mesh screen prevents the fiber from passingthrough, while the protein in the mixture passes through the screen,resulting in a separation of the fiber from the protein. In anembodiment, the fiber fraction is dried and can be used in dietary fiberproducts. In an embodiment, protein and carbohydrates are present in thefiber fraction.

In another embodiment, the pH of the mixture is adjusted to about 7 withthe addition of aqueous sodium hydroxide. In a further embodiment, theaqueous sodium hydroxide is a solution of about 1% to about 40%,optionally about 5% to about 30%, by weight of sodium hydroxide inwater.

In another embodiment, the mixture is optionally milled using a wetmilling process. In an embodiment, the wet milling of the mixtureresults in thorough mixing of the macroalgae and/or microalgae with themixing solvent. Thorough mixing of the mixture disperses the proteinparticles and releases natural sugar compounds that are trapped insidethe insoluble protein particles in the mixing solvent. In addition, themixing suspends the solids of the protein-enriched algae in the mixingsolvent.

In another embodiment of the present disclosure, the mixture iscentrifuged using a decanting centrifuge. In an embodiment, the mixtureis centrifuged with a decanting centrifuge at a speed of about 1000 rpmto about 2000 rpm. In another embodiment, the speed is about 1500 rpm.

In another embodiment, the protein slurry is then centrifuged using adisc stack centrifuge to separate insoluble proteins from solubleproteins, forming a protein precipitate and a soluble protein fraction.In an embodiment, the protein slurry is pumped to a disc centrifuge. Thecentrifuge has a bowl which spins at about 7500 rpm. As the slurryenters the centrifuge bowl, the slurry is brought up to the same speedas the bowl, which results in high centrifugal forces, about 6500 timesthe force of gravity acting on the mixture. The heavier proteinprecipitate is forced to the outside of the bowl. The soluble proteinfraction is forced towards the axis of the bowl. The heavy precipitatecollects around the outside of the bowl which are removed from the bowlperiodically or continuously. The protein slurry is fed to thecentrifuge continuously while the liquid soluble protein fraction ispumped out continuously. In an embodiment, the disc centrifuge operatesat a speed of about 6500 rpm to about 8500 rpm.

In a further embodiment, the protein precipitate is washed with anextraction solvent to purify the protein precipitate and dissolveresidual sugars and other non-desirable compounds. It will be understoodthat an extraction solvent will be any solvent which dissolves thecarbohydrates and other non-desirable compounds. In an embodiment, theextraction solvent is water, methanol, ethanol or isopropanol, andmixtures thereof. In another embodiment, the extraction solvent is wateror ethanol, and mixtures thereof. In another embodiment, the extractionsolvent is water. In an embodiment, the protein precipitate is washed atleast twice with the extraction solvent.

In another embodiment, the washed protein precipitate is thencentrifuged again with a disc stack centrifuge at a speed of about 6500rpm to about 8500 rpm to obtain a protein precipitate. In anotherembodiment, the washing extracts from the centrifugation are added tothe soluble protein fraction.

In another embodiment, the washed protein precipitate is dried to form aprotein concentrate comprising a protein content of about 30% to about90% on a dry weight basis. In a further embodiment, the washed proteinprecipitate is dried in a vacuum dryer, fluidized bed dryer or ringdryer to form the protein concentrate possessing a protein content ofabout 30% to less than 90%. It will be understood by a person skilled inthe art that the washed protein precipitate can be used as a proteinisolate without drying. However, the dried protein isolate has a bettershelf life as removal of the solvent, for example water, results in amore stable protein isolate.

In another embodiment of the present disclosure, there is provided aprocess for the production of a protein isolate comprising a proteincontent of greater than 90% on a dry weight basis. In an embodiment, ageneral process for the production of a protein isolate and hydrolyzedproteins having a protein content greater than 90% is illustrated inFIGS. 11-12.

Accordingly, a process for the production of a protein isolate frommacroalgae and/or microalgae is disclosed, comprising:

removing fiber from the defatted or protein-enriched macroalgae and/ormicroalgae, comprising:

-   -   i) mixing the macroalgae and/or microalgae with a mixing solvent        to form a mixture;        -   screening the mixture through a mesh screen of about 10 to            about 200 US mesh size to remove fiber,        -   optionally adjusting the pH of the mixture to a pH of about            7;        -   optionally milling the mixture; and        -   centrifuging the mixture to remove fiber,        -   and forming a protein slurry;    -   ii) centrifuging the protein slurry to form a protein        precipitate and a soluble protein fraction;    -   iii) filtering the soluble protein fraction; and    -   iv) drying the soluble protein to form the protein isolate.

In an embodiment, the soluble protein fraction is obtained using thesame process as described above.

It will be understood by a person skilled in the art that the steps ofthe process do not have to be followed exactly. For example, a personskilled in the art would recognize that the milling step could beperformed before the screening step.

In an embodiment of the disclosure, the mixing solvent comprises wateror a salt solution. In an embodiment, the salt solution comprises lessthan 5%, optionally about 3% to about 4%, or 3.5% by weight of salt insolution. In a further embodiment, the mixing solvent comprises water.In another embodiment, the ratio of macroalgae and/or microalgae to themixing solvent is about 1:3 to about 1:20. In a further embodiment, theratio is about 1:6 to about 1:10. In an embodiment, the ratio is about1:6 to about 1:8.

In an embodiment, the soluble protein fraction is purified byultrafiltration and diafiltration using a membrane filtration apparatus.In an embodiment, when ultrafiltration is utilized, the soluble proteinfraction is heated to a temperature of about 1° C. to about 60° C.,optionally 40° C. to about 55° C., before being passed through anultrafiltration apparatus fitted with membranes to filter proteinslarger than about 10,000 daltons, optionally about 30,000, or about100,000 daltons. The filtered protein is recycled back to the feed tankwhile the liquid is discarded. The ultrafiltration process is continueduntil the amount of protein that has been filtered in the feed tank isequal to about 30% to about 40% of its initial weight of the solubleprotein fraction.

In a further embodiment, when diafiltration is utilized, it is conductedat about 1° C. to about 60° C., optionally about 40° C. to about 55° C.,using the diafiltration unit, which is fitted with the membranes tofilter proteins larger than about 10,000 daltons, optionally about30,000, or about 100,000 daltons. The original volume of soluble proteinfraction in the feed tank is held constant by adding water to make upfor the removed liquid. The filtered protein is recycled back to thefeed tank. The amount of water added to maintain the original volume ofprotein solution is about 2 times the original volume of soluble proteinfraction. For example, if 100 L of soluble protein fraction is used, 200L of water is added to the soluble protein fraction in the feed tankduring the cycle of diafiltration. The volume of the feed tank is keptconstant at 100 L with the continued addition of water to the feed tankwhile the liquid is removed from the system through diafiltration.

In an embodiment, after the soluble protein fraction has been filtered,the filtered soluble protein is spray dried to form a high functionalprotein isolate comprising a protein content of greater than about 90%on a dry weight basis. It will be understood by a person skilled in theart that spray drying is the transformation of a feed from a fluid stateinto a dried form by spraying the feed into a circulating hot airmedium. Generally, spray drying transforms the filtered protein intomany droplets which are then exposed to a fast current of hot air. As aresult of the very large surface area of the droplets the water in theprotein evaporates almost instantaneously and the droplets aretransformed into powdery dry protein particles. In an embodiment, theinlet temperature is about 180° C. to about 220° C. which is thetemperature of the hot air entering the spray dryer chamber, the outlettemperature is about 75° C. to about 90° C., which is the temperature ofthe exhaust, and the feed temperature is about 40° C. to about 50° C. Itwill be understood by a person skilled in the art that the washedprotein precipitate can be used as a protein isolate without drying.However, the dried protein isolate has a better shelf life as removal ofthe solvent, for example water, results in a more stable proteinisolate.

In another embodiment of the present disclosure, there is provided aprocess for the production of a protein isolate which is subsequentlymodified or hydrolyzed to form a high functional protein isolate or amixture of hydrolyzed proteins, peptides and amino acids comprising aprotein content of greater than 90% on a dry weight basis.

Accordingly, in an embodiment of the present disclosure, a process forthe production of a protein isolate from macroalgae and/or microalgae isdisclosed, comprising:

-   -   removing fiber from the defatted or protein-enriched macroalgae        and/or microalgae, comprising:    -   i) mixing the macroalgae and/or microalgae with a mixing solvent        to form a mixture;        -   optionally screening the mixture through a mesh screen of            about 10 to about 200 US mesh size to remove fiber,        -   optionally adjusting the pH of the mixture to a pH of about            7;        -   optionally milling the mixture; and        -   centrifuging the mixture to remove fiber,        -   and forming a protein slurry;    -   ii) centrifuging the protein slurry to form a protein        precipitate and a soluble protein fraction;    -   iii) mixing the protein precipitate with water to form a protein        precipitate mixture and optionally adjusting the pH of the        mixture to a pH of about 3 to about 7;    -   iv) adding cellulase complex to the protein precipitate mixture        to hydrolyze residual fiber;    -   v) washing the protein precipitate with an extraction solvent        and centrifuging to form a protein isolate.

It will be understood by a person skilled in the art that the steps ofthe process do not have to be followed exactly. For example, a personskilled in the art would recognize that the milling step could beperformed before the screening step.

In another embodiment of the disclosure, the mixing solvent compriseswater or a salt solution. In an embodiment, the salt solution comprisesless than 5%, optionally about 3% to about 4%, or 3.5% by weight of saltin solution. In a further embodiment, the mixing solvent compriseswater. In another embodiment, the ratio of macroalgae and/or microalgaeto the mixing solvent is about 1:3 to about 1:20. In a furtherembodiment, the ratio is about 1:6 to about 1:10. In an embodiment, theratio is about 1:6 to about 1:8.

In a further embodiment, the mixture is wet screened resulting in aseparation of the fiber from the mixture which contains the protein. Inanother embodiment of the disclosure, the mesh screen comprises a USmesh screen of size about 20 to about 200 mesh. In an embodiment, themesh screen is of size 40 US mesh size. In a further embodiment, themesh screen is a vibratory screen. The mesh screen prevents the fiberfrom passing through, while the protein in the mixture passes throughthe screen, resulting in a separation of the fiber from the protein.This results in a mixture of protein which passes through the screen anda fiber fraction which is trapped by the screen. In an embodiment, thefiber fraction is dried and can be used in dietary fiber products. In anembodiment, some protein and carbohydrates are present in the fiberfraction.

In another embodiment, the pH of the mixture is optionally adjusted toabout 7 with the addition of aqueous sodium hydroxide. In a furtherembodiment, the aqueous sodium hydroxide is a solution of about 1% toabout 40%, optionally about 5% to about 30%, by weight of sodiumhydroxide in water.

In another embodiment, the mixture is optionally milled using a wetmilling process. In an embodiment, the wet milling of the mixtureresults in thorough mixing of the macroalgae and/or microalgae with themixing solvent.

In another embodiment of the present disclosure, the mixture iscentrifuged using a decanting centrifuge. In an embodiment, the mixtureis centrifuged with a decanting centrifuge at a speed of about 1000 rpmto about 2000 rpm. In another embodiment, the speed is about 1500 rpm.

In another embodiment, the protein slurry is centrifuged using a discstack centrifuge to separate insoluble proteins from soluble proteins,forming a protein precipitate and a soluble protein fraction. In anembodiment, the soluble protein fraction is filtered as described above.In an embodiment, the disc centrifuge operates continuously at a speedof about 6500 rpm to about 8500 rpm at a temperature of about 1° C. toabout 60° C., optionally about 20° C. to about 40° C., or optionally atabout 20° C. to about 25° C.

In an embodiment of the disclosure, the precipitated protein is mixedwith water and its pH optionally adjusted for the addition of cellulasecomplex, in a similar manner as described above. This additionalenzymatic step hydrolyzes residual fiber and allows the removal of fiberfrom the protein precipitate.

In another embodiment, after treatment with the cellulase complex, theprotein precipitate is washed at least once with an extraction solventto remove water-soluble sugar compounds as a result of the fiberhydrolyzation by the cellulase complex. In an embodiment, the extractionsolvent is water. In an embodiment, the protein precipitate mixture iswashed at least twice with the extraction solvent. In an embodiment, theratio of extraction solvent to the precipitated protein is about 10:1 toabout 1:1, optionally about 4:1 to about 2:1. The mixture is thenfurther centrifuged to obtain a protein precipitate that has beenfurther purified.

In an embodiment, the further purified protein precipitate is thensubjected to high pressure jet cooking to obtain a high functionalprotein isolate having a protein content of greater than about 90% on adry weight basis. In an embodiment, the jet cooking of the proteinisolate occurs at a temperature of about 90° C. to about 120° C. forabout 1 second to about 2 minutes, optionally about 3 seconds to about30 seconds. As will be understood by a person skilled in the art, jetcooking involves the injection of steam into the purified protein, andresults in the pasteurization of the protein and improves the functionalproperties of the protein isolate.

In another embodiment, the further purified protein precipitate ishydrolyzed using proteases to form a hydrolyzed protein extractcontaining hydrolyzed proteins, peptides and amino acids having aprotein content of greater than about 90% on a dry weight basis. In anembodiment, the proteases are, for example, Alcalase® and Flavourzyme®.Alcalase® and Flavourzyme® were obtained from Novozymes North America,Inc., Franklinton, N.C. USA. This step hydrolyzes the protein in theprotein precipitate into smaller peptides and amino acids, which aresoluble in nutritional drinks and are easily adsorbed. In an embodiment,the purified protein precipitate is mixed with water to form a proteinslurry, which is optionally followed by pH adjustment to a pH of about6.0 to about 10.0, optionally about 7.5 to about 8.5. In an embodiment,the Alcalase® is added in a ratio of about 0.5% based on the dry weightof the protein slurry. In a further embodiment, the temperature isadjusted to about 20° C. to about 65° C., optionally about 50° C. toabout 60° C., or about 60° C., for about 1 to about 4 hours. Thehydrolyzed protein slurry is then cooled to about 30° C. to about 50°C., or about 40° C. to about 50° C., or about 50° C. The pH of themixture is then adjusted to a pH of about 5.0 to about 7.0, or about 6.0to about 7.0, or about 6.5, and a protease to form a hydrolyzed proteinextract, such as Flavourzyme®, is then added to the mixture. In anembodiment, the protease to form a hydrolyzed protein extract, such asFlavourzyme®, is added in a ratio of about 0.5% based on the dry weightof the protein slurry. In a further embodiment, the mixture is thenheated to a temperature of about 20° C. to about 60° C., optionallyabout 40° C. to about 60° C., or about 45° C. to about 55° C., for about1 to about 4 hours. The hydrolyzed protein mixture is then centrifugedto separate the hydrolyzed protein extract from the insoluble solids.The soluble hydrolyzed protein extract is then spray dried as describedabove, while the extract from the centrifugation is added to the solubleprotein fraction as described above.

In another embodiment of the disclosure, there is a provided a processfor the production of a protein concentrate from a macroalgae and/ormicroalgaeas shown in FIGS. 13-19, comprising:

-   -   i) mixing the macroalgae and/or microalgae with a mixing solvent        to form a mixture;    -   ii) optionally adjusting the pH of the mixture to a pH of about        2.0 to about 10.0;    -   iii) separating fiber from the mixture to form a protein slurry,        wherein the protein slurry comprises a soluble protein fraction        and an insoluble protein fraction;    -   iv) optionally repeating steps i)-iii) by mixing the protein        slurry with additional partially defatted, fully defatted or        protein-enriched algae;    -   v) mixing the protein slurry with an extraction solvent to form        an extract and a washed insoluble protein fraction;    -   vi) separating the extract from the washed insoluble protein        fraction;    -   vii) optionally repeating steps v) and vi) at least once; and    -   viii) desolventizing the washed insoluble protein fraction to        form a protein concentrate.

In another embodiment of the disclosure, the ratio of macroalgae and/ormicroalgae to mixing solvent is about 1:3 to about 1:30 (w/w). Inanother embodiment, the ratio of macroalgae and/or microalgae to solventis about 1:5 to about 1:20 (w/w). In a further embodiment, the ratio isabout 1:6 to about 1:12 (w/w). In an embodiment, the ratio is about 1:8to about 1:10 (w/w).

In a further embodiment of the disclosure, the mixing solvent compriseswater or an aqueous solution comprising a polysaccharide, a salt or analcohol. In an embodiment, the mixing solvent is water. In anotherembodiment, the polysaccharide is guar gum.

In an embodiment, the pH of the protein slurry is adjusted to a pH ofabout 6.5 to about 10.0. In a further embodiment, the pH of the proteinslurry is adjusted to a pH of about 7.0 to about 9.0.

In an embodiment, the mixture is centrifuged to separate the fiber frommixture and form the protein slurry. In an embodiment, the mixture iscentrifuged at a speed of about 1,000 rpm to about 2,000 rpm. In afurther embodiment, the mixture is centrifuged at a speed of about 1,400to about 1,600 rpm. In an embodiment, the mixture is centrifuged using adecanter centrifuge.

The centrifugation of the mixture results in three layers: i) aninsoluble fiber layer and a protein slurry on top of the fiber, whichwas comprised of ii) an insoluble protein fraction and iii) a solubleprotein fraction. Separation of the top and middle layers (the solubleprotein extract and the insoluble fine protein fraction) from the bottomlayer (coarse fiber solids), resulted in the protein slurry with fiberremoved. In an embodiment, the bird decanter was operated at a low pooldepth with a bowl speed of between about 1,000 rpm and about 2,000 rpm,optionally 1,400 to about 1,600 rpm, suitably about 1,500 rpm. It wasdetermined that when the speed of the centrifugation is too high, forexample at 5,000 rpm, the insoluble protein fraction settles with thefiber. If the speed of the centrifugation is too low, fiber will remainin the protein slurry. Accordingly, in an embodiment, when the speed ofthe centrifugation is between about 1,000 rpm and about 2,000 rpm,optionally 1,400 to about 1,600 rpm, suitably about 1,500 rpm, the fiberin the mixture is separated from both the soluble and insoluble protein.

In another embodiment of the disclosure, mixing the protein slurry withadditional macroalgae and/or microalgae repeated at least once. In afurther embodiment, mixing the protein slurry with additional macroalgaeand/or microalgae is repeated at least two to seven times. In anembodiment, recycling the protein slurry with additional macroalgaeand/or microalgae increases the solid content of the algae beingprocessed, and accordingly, reduces the overall processing volume.

In an embodiment of the disclosure, the extraction solvent compriseswater, methanol, ethanol, isopropanol, or mixtures thereof. In anembodiment, the extraction solvent comprises ethanol. In anotherembodiment, the extraction solvent comprises at least about 50% ethanol.In an embodiment, the extraction solvent comprises at least about 70%ethanol. In a further embodiment, the extraction solvent comprises atleast about 90% ethanol.

In a further embodiment, the extract is separated from the washedinsoluble protein fraction using centrifugation, vacuum filtration,pressure filtration, decantation or gravity draining. In an embodiment,the extract is separated from the washed insoluble protein fractionusing centrifugation.

In another embodiment of the disclosure, wherein steps iv) and v) arerepeated at least twice.

In a further embodiment, the process further comprises the step ofdrying the washed insoluble protein fraction to form the proteinconcentrate. In an embodiment, the protein concentrate is dried in avacuum dryer, fluidized bed dryer, hot air dryer ring dryer or spraydryer.

In an embodiment, the protein concentrate comprises a protein content ofabout 30% to about 90% on a dry weight basis.

In another embodiment of the disclosure, there is also provided aprocess for the production of a protein isolate from a partiallydefatted, fully defatted or protein enriched algae, comprising:

-   -   i) mixing the macroalgae and/or microalgae with alkaline water        to form a mixture;    -   ii) optionally adjusting the pH of the mixture to a pH of about        6.0 to about 10.0;    -   iii) separating fiber from the mixture to form a first protein        slurry, wherein the first protein slurry comprises a soluble        protein fraction and an insoluble protein fraction;    -   iv) separating the first protein slurry to form a protein solids        fraction and a soluble protein fraction;    -   v) mixing the protein solids fraction with water to form a        second protein slurry;    -   vi) separating the second protein slurry to form a second        protein solids fraction and a second soluble protein fraction;    -   vii) optionally repeating steps v) and vi) at least once;    -   viii) separating the soluble protein fractions to form a        clarified soluble protein fraction and a residual insoluble        protein fraction;    -   ix) optionally adjusting the pH of the clarified soluble protein        fraction to a pH of about 7;    -   x) separating the clarified soluble protein fraction, optionally        by filtering the clarified soluble protein fraction by membrane        filtration; and    -   xi) optionally drying the clarified soluble protein fraction.

In another embodiment of the disclosure, the ratio of macroalgae and/ormicroalgae to alkaline water is about 1:3 to about 1:30 (w/w). Inanother embodiment, the ratio of macroalgae and/or microalgae toalkaline water is about 1:5 to about 1:20 (w/w). In a furtherembodiment, the ratio is about 1:6 to about 1:12 (w/w). In anembodiment, the ratio is about 1:8 to about 1:10 (w/w).

In an embodiment of the disclosure, the pH of the alkaline water isabout 7 to about 12. In another embodiment, the pH of the first proteinslurry is adjusted to about 8.0 to about 9.5. In a further embodiment,the pH of the first protein slurry is adjusted to about 8.5 to about9.0.

In an embodiment, the mixture is centrifuged to separate the fiber fromthe protein slurry and form the protein extract. In an embodiment, themixture is centrifuged at a speed of about 1,000 rpm to about 2,000 rpm.In a further embodiment, the mixture is centrifuged at a speed of about1,400 to about 1,600 rpm. In an embodiment, the mixture is centrifugedusing a decanter centrifuge.

In another embodiment, the first protein slurry is centrifuged toseparate the protein solids fraction from the soluble protein fraction.In a further embodiment, the first protein slurry is centrifuged at aspeed of about 4,000 rpm to about 8,500 rpm. In a further embodiment,the first protein slurry is centrifuged at a speed of about 5,000 toabout 8,500 rpm.

In another embodiment of the disclosure, the ratio of the protein solidsfraction to water is about 1.0:0.5 to about 1.0:3.0 (w/w). In a furtherembodiment, the ratio of the protein solids fraction to water is about1.0:1.0 to about 1.0:2.0 (w/w).

In an embodiment, the soluble protein fractions are centrifuged to formthe clarified soluble protein fraction and the residual insolubleprotein fraction. In an embodiment, the soluble protein fractions arecentrifuged using a disc stack centrifuge at a speed of about 7,000 rpmto about 10,000 rpm. In a further embodiment, the soluble proteinfractions are centrifuged using a disc stack centrifuge at a speed ofabout 8,400 rpm to about 8,600 rpm.

In another embodiment of the disclosure, the pH of the clarified solubleprotein fraction is adjusted with alkali. In a further embodiment, thepH of the clarified soluble protein fraction is adjusted with sodiumhydroxide.

In an embodiment, the clarified soluble protein fraction is filteredusing an ultrafiltration apparatus. In a further embodiment, theultrafiltration apparatus comprises a membrane to filter proteins largerthan about 10,000 daltons. In another embodiment, the separation of theclarified soluble protein fraction is accomplished by adjusting the pHof the solution to the isoelectric point of the proteins (about pH of4.5), and consequently, the proteins are precipitated out of solution.In another embodiment, the proteins are cooked to precipitate theproteins from solution.

In another embodiment of the disclosure, the process further comprisesthe step of filtering the clarified soluble protein fraction using adiafiltration apparatus.

In another embodiment, the clarified soluble protein fraction is driedin a vacuum dryer, fluidized bed dryer, freeze dryer, ring dryer orspray dryer to form the protein isolate.

In another embodiment of the disclosure, the protein concentratecomprises a protein content of greater than about 90% on a dry weightbasis.

In another embodiment of the disclosure, there is also provided aprocess for the production of a hydrolyzed protein concentrate from apartially defatted, fully defatted or protein-enriched algae,comprising:

-   -   i) mixing the macroalgae and/or microalgae with water to form a        mixture;    -   ii) optionally adjusting the pH of the mixture to a pH of about        6.0 to about 10.0;    -   iii) separating the mixture to remove fiber from the mixture and        form a first protein slurry, wherein the first protein slurry        comprises a soluble protein fraction and an insoluble protein        fraction;    -   iv) optionally adjusting the pH of the first protein slurry to a        pH of about 7.0;    -   v) separating the first protein slurry to form a first protein        solids fraction and a first soluble protein fraction;    -   vi) mixing the first protein solids fraction with water to form        a second protein slurry;    -   vii) separating the second protein slurry to form a second        protein solids fraction and a second soluble protein fraction;    -   viii) mixing the second protein solids fraction with water to        form a third protein slurry;    -   ix) adjusting the pH of the third protein slurry to a pH of        about 7.0 to about 9.0;    -   x) mixing the third protein slurry with at least one protease to        form a hydrolyzed protein extract;    -   xi) separating the hydrolyzed protein extract from the third        protein slurry to form the hydrolyzed protein concentrate.

In another embodiment of the disclosure, the ratio of macroalgae and/ormicroalgae to water is about 1:3 to about 1:30 (w/w). In anotherembodiment, the ratio of macroalgae and/or microalgae to water is about1:5 to about 1:20 (w/w). In a further embodiment, the ratio is about 1:6to about 1:12 (w/w). In an embodiment, the ratio is about 1:8 to about1:10 (w/w).

In another embodiment, the pH of the mixture is adjusted to about 8.0 toabout 9.0. In a further embodiment, the pH of the mixture is adjusted toabout 8.5 to about 9.0.

In an embodiment, the mixture is centrifuged to separate the fiber fromthe protein slurry and form the protein extract. In an embodiment, themixture is centrifuged at a speed of about 1,000 rpm to about 2,000 rpm.In a further embodiment, the mixture is centrifuged at a speed of about1,400 to about 1,600 rpm. In an embodiment, the mixture is centrifugedusing a decanter centrifuge.

In another embodiment, the first protein slurry is centrifuged toseparate the protein solids fraction from the soluble protein fraction.In a further embodiment, the first protein slurry is centrifuged at aspeed of about 4,000 rpm to about 8,000 rpm. In a further embodiment,the first protein slurry is centrifuged at a speed of about 5,000 toabout 8,500 rpm.

In another embodiment, the ratio of the first and second protein solidsfraction to water is about 1.0:0.5 to about 1.0:3.0 (w/w). In a furtherembodiment, the ratio of the first and second protein solids fraction towater is about 1.0:1.0 to about 1.0:2.0 (w/w).

In another embodiment of the disclosure, the pH of the third proteinslurry is adjusted to about 8.0 to about 8.5.

In an embodiment of the disclosure, the ratio of the third proteinslurry to the protease is about 100:1 to about 5000:1 (w/w).

In an embodiment of the disclosure, the third protein slurry is mixedwith a protease at a temperature of about 50° C. to about 70° C. Inanother embodiment, the third protein slurry is mixed with a protease ata temperature of about 55 to about 65° C.

In another embodiment, the at least one protease comprises a proteasefrom Bacillus Licheniformis.

In a further embodiment, the process further comprises the step ofmixing the third protein slurry with a second protease.

In an embodiment, the ratio of the third protein slurry to the secondprotease is about 100:1 to about 5000:1 (w/w).

In another embodiment, the third protein slurry is mixed with the secondprotease at a temperature of about 40° C. to about 60° C. In anembodiment, the third protein slurry is mixed with the second proteaseat a temperature of about 45° C. to about 55° C.

In a further embodiment, the second protease comprises a fungalprotease/peptidase complex from Aspergillus oryzae.

In an embodiment, the hydrolyzed protein extract is separated using acentrifuge. In a further embodiment, the hydrolyzed protein extract isseparated using a decanter centrifuge at a speed of about 3,800 to about5,200 rpm.

In another embodiment, the clarified soluble protein fraction is driedin a vacuum dryer, fluidized bed dryer, ring dryer or spray dryer toform the protein isolate.

In a further embodiment, the hydrolyzed protein concentrate comprises aprotein content of greater than about 30% on a dry weight basis.

In an embodiment, the use of an extraction solvent, such as ethanol,leads to a protein concentrate or protein isolate having superiororganoleptic properties, as well as superior protein solubilityproperties, which therefore possesses better functional properties. Inan embodiment, the use of an extraction solvent, such as ethanol,results in the protein concentrates containing fewer impurities.Consequently, the protein concentrates are generally of higher qualityand have better functional properties.

In another embodiment of the disclosure, there is also provided aprocess for the production of a protein isolate from macroalgae and/ormicroalgae as shown in FIGS. 20-25, comprising:

-   -   i) mixing the macroalgae and/or microalgae with a blending        solvent, optionally water or alkaline water, to form a mixture        and optionally treating the mixture with phytase at a        temperature and a pH suitable for phytase activity;    -   ii) optionally adjusting the pH of the mixture to a pH of about        7.0 to about 10.0;    -   iii) separating fiber from the mixture to form a first protein        slurry, wherein the first protein slurry comprises a soluble        protein fraction and an insoluble protein fraction;    -   iv) separating the first protein slurry to form a protein solids        fraction and a soluble protein fraction;    -   v) optionally mixing the protein solids fraction with a second        blending solvent, optionally water, to form a second protein        slurry;    -   vi) optionally separating the second protein slurry to form a        second protein solids fraction and a second soluble protein        fraction;    -   vii) optionally repeating steps v) and vi) at least once;    -   viii) separating the soluble protein fractions to form a        clarified soluble protein fraction and a residual insoluble        protein fraction;    -   ix) optionally adjusting the pH of the clarified soluble protein        fraction to a pH of about 6 to about 9;    -   x) separating the clarified soluble protein fraction, optionally        by filtering the clarified soluble protein fraction by membrane        filtration; and    -   xi) optionally drying the clarified soluble protein fraction.

In another embodiment of the disclosure, the ratio of macroalgae and/ormicroalgae to water or alkaline water is about 1:4 to about 1:30 (w/w).In another embodiment, the ratio of macroalgae and/or microalgae towater or alkaline water is about 1:5 to about 1:20 (w/w). In a furtherembodiment, the ratio is about 1:6 to about 1:12 (w/w). In anembodiment, the ratio is about 1:8 to about 1:10 (w/w).

In an embodiment of the disclosure, the pH of the alkaline water isabout 7 to about 12. In another embodiment, the pH of the first proteinslurry is adjusted to about 8.0 to about 9.5. In a further embodiment,the pH of the first protein slurry is adjusted to about 8.5 to about9.0.

In another embodiment of the disclosure, the mixture is separated bycentrifugation, gravity sedimentation, a gravity table or hydrocycloneto separate the fiber from the mixture and form the protein slurry. In afurther embodiment, the mixture is separated by centrifugation toseparate the fiber from the mixture and form the protein slurry. In anembodiment, the mixture is centrifuged at a speed of about 1,000 rpm toabout 2,000 rpm. In a further embodiment, the mixture is centrifuged ata speed of about 1,400 to about 1,600 rpm. In an embodiment, the mixtureis centrifuged using a decanter centrifuge.

In another embodiment, the first protein slurry is centrifuged,optionally using a disc stack centrifuge, to separate the protein solidsfraction from the soluble protein fraction. In a further embodiment, thefirst protein slurry is centrifuged at a speed of about 4,000 rpm toabout 8,000 rpm. In a further embodiment, the first protein slurry iscentrifuged at a speed of about 6,500 to about 7,500 rpm.

In another embodiment of the disclosure, the ratio of the protein solidsfraction to water is about 1.0:0.5 to about 1.0:3.0 (w/w). In a furtherembodiment, the ratio of the protein solids fraction to water is about1.0:1.0 to about 1.0:2.0 (w/w).

In an embodiment, the soluble protein fractions are centrifuged to formthe clarified soluble protein fraction and the residual insolubleprotein fraction. In an embodiment, the soluble protein fractions arecentrifuged using a disc stack centrifuge at a speed of about 7,000 rpmto about 10,000 rpm. In a further embodiment, the soluble proteinfractions are centrifuged using a disc stack centrifuge at a speed ofabout 7,500 rpm to about 8,500 rpm.

In another embodiment of the disclosure, the pH of the clarified solubleprotein fraction is adjusted with alkali. In a further embodiment, thepH of the clarified soluble protein fraction is adjusted with sodiumhydroxide.

In an embodiment, the clarified soluble protein fraction is filteredusing an ultrafiltration apparatus. In a further embodiment, theultrafiltration apparatus comprises a membrane to filter proteins largerthan about 10,000 daltons.

In another embodiment of the disclosure, the process further comprisesthe step of filtering the clarified soluble protein fraction using adiafiltration apparatus.

In another embodiment, the clarified soluble protein fraction is driedin a vacuum dryer, fluidized bed dryer, ring dryer or spray dryer toform the protein isolate.

In another embodiment, the protein isolate comprises a hydrolyzedprotein isolate. In another embodiment, the protein isolate ishydrolyzed to produce peptides and free amino acids. In anotherembodiment, the hydrolyzed protein isolate comprises peptides and/orfree amino acids.

In another embodiment of the disclosure, the protein isolate comprises aprotein content of greater than about 90% on a dry weight basis.

In another embodiment of the disclosure, there is also provided aprocess for the production of a hydrolyzed protein concentrate frommacroalgae and/or microalgae as shown in FIGS. 25-26, comprising:

-   -   i) mixing the macroalgae and/or microalgae with a blending        solvent, optionally water, to form a first mixture and        optionally treating the mixture with phytase at a temperature        and a pH suitable for phytase activity;    -   ii) optionally adjusting the pH of the first mixture to a pH of        about 6.5 to about 10.0;    -   iii) separating the first mixture to remove fiber from the first        mixture and form a protein slurry and an insoluble fiber        fraction, wherein the protein slurry comprises a soluble protein        fraction and an insoluble protein fraction and the insoluble        fiber fraction comprises insoluble fiber and a second insoluble        protein fraction;    -   iv) optionally mixing the insoluble fiber fraction with a second        blending solvent, optionally water, to form a washed insoluble        fiber fraction and an extract;    -   v) separating the washed insoluble fiber fraction from the        extract;    -   vi) optionally mixing the washed insoluble fiber fraction with a        blending solvent, optionally water, to form a second mixture;    -   vii) optionally adjusting the pH of the second mixture to a pH        suitable for enzymatic activity;    -   viii) mixing the second mixture with at least one protease to        form a hydrolyzed protein extract;    -   ix) separating the hydrolyzed protein extract from the second        mixture to form the hydrolyzed protein concentrate and a second        insoluble fiber fraction; and    -   x) optionally drying the hydrolyzed protein concentrate.

In another embodiment of the disclosure, the ratio of macroalgae and/ormicroalgae to water is about 1:4 to about 1:30 (w/w). In anotherembodiment, the ratio of macroalgae and/or microalgae to water is about1:5 to about 1:20 (w/w). In a further embodiment, the ratio is about 1:6to about 1:12 (w/w). In an embodiment, the ratio is about 1:8 to about1:10 (w/w).

In another embodiment, the pH of the first mixture is adjusted to about8.0 to about 9.5. In a further embodiment, the pH of the first mixtureis adjusted to about 8.5 to about 9.0.

In another embodiment of the disclosure, the first mixture is separatedby centrifugation, gravity sedimentation, a gravity table orhydrocyclone to separate the fiber from the first mixture and form theprotein slurry. In a further embodiment, the mixture is separated bycentrifugation to separate the fiber from the mixture and form theprotein slurry. In an embodiment, the first mixture is centrifuged at aspeed of about 1,000 rpm to about 2,000 rpm. In a further embodiment,the first mixture is centrifuged at a speed of about 1,400 to about1,600 rpm. In an embodiment, the mixture is centrifuged using a decantercentrifuge.

In another embodiment, the ratio of the insoluble fiber fraction orwashed insoluble fiber fraction to water is about 1.0:0.5 to about1.0:3.0 (w/w). In a further embodiment, the ratio of the insoluble fiberfraction or washed insoluble fiber fraction to water is about 1.0:1.0 toabout 1.0:2.0 (w/w).

In another embodiment, the washed insoluble fiber fraction iscentrifuged to separate the washed insoluble fiber fraction fromextract. In a further embodiment, the washed insoluble fiber fraction iscentrifuged at a speed of about 2,000 rpm to about 6,000 rpm. In afurther embodiment, washed insoluble fiber fraction is centrifuged at aspeed of about 3,000 to about 5,500 rpm.

In another embodiment of the disclosure, the pH of the second mixture isadjusted to about 8.0 to about 9.0.

In an embodiment of the disclosure, the ratio of the second mixture tothe protease is about 100:1 to about 5000:1 (w/w).

In an embodiment of the disclosure, the second mixture is mixed with aprotease at a temperature of about 40° C. to about 60° C. In anotherembodiment, the second mixture is mixed with a protease at a temperatureof about 45° C. to about 55° C.

In another embodiment, the at least one protease comprises a proteasefrom Bacillus Licheniformis.

In a further embodiment, the process further comprises the step ofmixing the second mixture with a second protease.

In an embodiment, the ratio of the second mixture to the second proteaseis about 250:1 to about 5000:1 (w/w).

In another embodiment, the second mixture is mixed with the secondprotease at a temperature of about 50° C. to about 70° C. In anembodiment, the second mixture is mixed with the second protease at atemperature of about 55° C. to about 65° C.

In a further embodiment, the second protease comprises a fungalprotease/peptidase complex from Aspergillus oryzae.

In another embodiment, the hydrolyzed protein concentrate is dried in avacuum dryer, fluidized bed dryer, ring dryer or spray dryer to form theprotein isolate.

In a further embodiment, the hydrolyzed protein concentrate comprises aprotein content of about 50% to about 90% on a dry weight basis.

In another embodiment, the process further comprises mixing thehydrolyzed protein extract with water to form a third mixture. In afurther embodiment, the process further comprises filtering the thirdmixture fraction and the filtering comprises ultrafiltration. In anembodiment, the ultrafiltration comprises contacting the third mixturewith an ultrafiltration apparatus that comprises a membrane to filterproteins larger than about 1,000 daltons.

In another embodiment, the process further comprises mixing the secondinsoluble fiber fraction to form a washed hydrolyzed protein extract anda washed second insoluble fiber fraction and separating the form thewashed hydrolyzed protein extract from the washed second insoluble fiberfraction. In another embodiment, the washed hydrolyzed protein extractis combined with the hydrolyzed protein extract.

In an embodiment of the disclosure, there is also provided a process forthe production of a protein concentrate from macroalgae and/ormicroalgae as shown in FIGS. 27-31, comprising:

-   -   i) mixing the macroalgae and/or microalgae with a blending        solvent, optionally water, a saline solution or a polysaccharide        solution, to form a mixture and optionally treating the mixture        with phytase at a temperature and a pH suitable for phytase        activity;    -   ii) optionally adjusting the pH of the mixture to a pH of about        2.0 to about 10.0;    -   iii) separating fiber from the mixture to form a protein slurry,        wherein the protein slurry comprises a first soluble protein        fraction and an insoluble protein fraction;    -   iv) optionally repeating steps i)-iii) by mixing the protein        slurry with additional algae;    -   v) separating the soluble protein fraction from the insoluble        protein fraction;    -   vi) washing the insoluble protein fraction with a second        blending solvent, optionally water, saline solution or        polysaccharide solution, to form a washed insoluble protein        fraction and a second soluble protein fraction;    -   vii) separating the washed insoluble protein fraction and the        second soluble protein fraction;    -   viii) combining and separating the first and second soluble        protein fractions to form a protein concentrate, optionally by        filtering the first and second soluble protein fractions to form        a protein concentrate or isolate;    -   ix) combining the washed insoluble protein fraction with the        protein concentrate to form a combined protein concentrate or        isolate; and    -   x) optionally drying the combined protein concentrate.

In another embodiment of the disclosure, the ratio of macroalgae and/ormicroalgae to water is about 1:3 to about 1:30 (w/w). In anotherembodiment, the ratio of macroalgae and/or microalgae to water is about1:5 to about 1:20 (w/w). In a further embodiment, the ratio is about 1:6to about 1:12 (w/w). In an embodiment, the ratio is about 1:8 to about1:10 (w/w).

In an embodiment, the pH of the mixture is adjusted to a pH of about 6.5to about 10.0. In another embodiment, the pH of the mixture is adjustedto a pH of about 7.0 to about 9.0.

In another embodiment of the disclosure, the mixture is separated bycentrifugation, gravity sedimentation, a gravity table or hydrocycloneto separate the fiber from the mixture and form the protein slurry. In afurther embodiment, the mixture is separated by centrifugation toseparate the fiber from the mixture and form the protein slurry. In anembodiment, the mixture is centrifuged at a speed of about 1,000 rpm toabout 2,000 rpm. In a further embodiment, the mixture is centrifuged ata speed of about 1,400 to about 1,600 rpm. In an embodiment, the mixtureis centrifuged using a decanter centrifuge.

In another embodiment, the protein slurry is centrifuged to separate theprotein solids fraction from the soluble protein fraction. In anembodiment, the protein slurry is centrifuged at a speed of about 6,000rpm to about 8,500 rpm in a disc stack centrifuge. In anotherembodiment, the protein slurry is centrifuged at a speed of about 6,500to about 7,500 rpm.

In another embodiment, the ratio of the insoluble protein fraction towater is about 1.0:0.5 to about 1.0:3.0 (w/w). In a further embodiment,the ratio of the insoluble protein fraction to water is about 1.0:1.0 toabout 1.0:2.0 (w/w).

In another embodiment, the washed insoluble protein fraction and thesecond soluble protein fraction are separated using a centrifuge. In anembodiment, the washed insoluble protein fraction and the second solubleprotein fraction are centrifuged at a speed of about 6,000 rpm to about8,500 rpm in a disc stack centrifuge. In a further embodiment, thewashed insoluble protein fraction and the second soluble proteinfraction are centrifuged at a speed of about 6,500 to about 7,500 rpm.

In another embodiment, the first and second soluble protein fractionsare filtered using an ultrafiltration apparatus. In a furtherembodiment, the ultrafiltration apparatus comprises a membrane to filterproteins larger than about 10,000 daltons. In an embodiment, the processfurther comprises the step of filtering the first and second solubleprotein fractions using a diafiltration apparatus.

In another embodiment, the combined protein concentrate is dried in avacuum dryer, fluidized bed dryer, ring dryer or spray dryer to form thedried protein concentrate.

In another embodiment, the protein concentrate comprises a hydrolyzedprotein concentrate. In another embodiment, the protein concentrate ishydrolyzed to produce peptides and free amino acids. In anotherembodiment, the hydrolyzed protein concentrate comprises peptides and/orfree amino acids.

In a further embodiment, the protein concentrate comprises a proteincontent of about 30% to about 90% on a dry weight basis.

In an embodiment of the disclosure, there is also provided a process forthe production of a protein isolate from macroalgae and/or microalgaecomprising:

-   -   i) mixing the macroalgae and/or microalgae with a blending        solvent, optionally water, to form a mixture and optionally        treating the mixture with phytase at a temperature and a pH        suitable for phytase activity;    -   ii) optionally adjusting the pH of the mixture to a pH of about        2.0 to about 10.0;    -   iii) separating fiber from the mixture to form a protein slurry,        wherein the protein slurry comprises a soluble protein fraction        and an insoluble protein fraction;    -   iv) washing the fiber with a second blending solvent, optionally        water, to form a washed fiber fraction;    -   vi) separating the washed fiber fraction to form a second        protein slurry and washed fiber solids;    -   vii) combining and separating the first and second protein        slurries to form a protein concentrate, optionally by filtering        the first and second soluble protein fractions to form a protein        concentrate; and    -   ix) optionally drying the protein concentrate.

In another embodiment of the disclosure, the ratio of macroalgae and/ormicroalgae to water is about 1:3 to about 1:30 (w/w). In anotherembodiment, the ratio of macroalgae and/or microalgae to water is about1:5 to about 1:20 (w/w). In a further embodiment, the ratio is about 1:6to about 1:12 (w/w). In an embodiment, the ratio is about 1:8 to about1:10 (w/w).

In an embodiment, the pH of the mixture is adjusted to a pH of about 6.5to about 10.0. In another embodiment, the pH of the mixture is adjustedto a pH of about 7.0 to about 9.0.

In another embodiment of the disclosure, the mixture is separated bycentrifugation, gravity sedimentation, a gravity table or hydrocycloneto separate the fiber from the mixture and form the protein slurry. In afurther embodiment, the mixture is separated by centrifugation toseparate the fiber from the mixture and form the protein slurry. In anembodiment, the mixture is centrifuged at a speed of about 1,000 rpm toabout 2,000 rpm. In a further embodiment, the mixture is centrifuged ata speed of about 1,400 to about 1,600 rpm. In an embodiment, the mixtureis centrifuged using a decanter centrifuge.

In another embodiment, the ratio of the fiber fraction to water is about1.0:0.5 to about 1.0:3.0 (w/w). In a further embodiment, the ratio ofthe insoluble protein fraction to water is about 1.0:1.0 to about1.0:2.0 (w/w).

In another embodiment of the disclosure, the washed fiber fraction isseparated by centrifugation, gravity sedimentation, a gravity table orhydrocyclone to separate the fiber solids and form second the proteinslurry. In a further embodiment, the washed fiber fraction is separatedby centrifugation to separate the fiber and form the second proteinslurry. In an embodiment, the mixture is centrifuged at a speed of about1,000 rpm to about 2,000 rpm. In a further embodiment, the fiberfraction is centrifuged at a speed of about 1,400 to about 1,600 rpm. Inan embodiment, the fiber fraction is centrifuged using a decantercentrifuge.

In another embodiment, the first and second slurries are filtered usingan ultrafiltration/microfiltration apparatus. In a further embodiment,the ultrafiltration/microfiltration apparatus comprises a membrane tofilter proteins larger than about 10,000 daltons. In an embodiment, theprocess further comprises the step of filtering the first and secondslurries using a diafiltration apparatus.

In another embodiment, the protein concentrate is dried in a vacuumdryer, fluidized bed dryer, ring dryer or spray dryer to form the driedprotein concentrate.

In another embodiment, the protein isolate comprises a hydrolyzedprotein isolate. In another embodiment, the protein isolate ishydrolyzed to produce peptides and free amino acids. In anotherembodiment, the hydrolyzed protein isolate comprises peptides and/orfree amino acids.

In a further embodiment, the protein concentrate comprises a proteincontent of about 30% to about 90% on a dry weight basis.

The present disclosure also relates to processes for the production ofprotein concentrates and protein isolates, in which the macroalgaeand/or microalgae is subjected to low g-forces to separate the fiberfrom the insoluble and soluble protein fractions. Removing the fiberfrom a protein mixture using low g-forces, separates the insoluble fiberfrom the protein fraction, and in particular the insoluble proteinfraction, which consequently increases the amount of recoverable proteinfrom macroalgae and/or microalgae.

The present disclosure relates to processes for the production ofprotein concentrates and protein isolates, in which the macroalgaeand/or microalgae is subjected to low g-forces to separate the fiberfrom the insoluble and soluble protein fractions. Removing the fiberfrom a protein mixture using low g-forces, separates the insoluble fiberfrom the protein fraction, and in particular the insoluble proteinfraction, which consequently increases the amount of recoverable proteinfrom macroalgae and/or microalgae.

Accordingly, the present disclosure includes a process for theproduction of a protein concentrate from macroalgae and/or microalgae asshown in FIG. 32, comprising:

i) mixing the macroalgae and/or microalgae with a first blending solventto form a mixture;

ii) optionally treating the mixture with phytase at a temperature and apH suitable for phytase activity;

iii) optionally adjusting the pH of the mixture to a pH between 6.0 and10.0;

iv) subjecting the mixture to a g-force sufficient to separate themixture to form

-   -   a) a fiber fraction, and    -   b) protein fractions comprising an insoluble protein fraction        and a soluble protein fraction;

v) optionally mixing the fiber fraction with a second blending solventand repeating step iv);

vi) optionally adjusting the pH of the protein fraction to a pH between4.0 and 6.0;

vii) heating the protein fraction to a temperature between 80° C. and100° C. to precipitate the proteins; and

viii) separating the precipitated proteins from the protein fraction toform the protein concentrate.

In another embodiment, the first and second blending solvents comprisewater, a saline solution or a polysaccharide solution. In a furtherembodiment, the first and second blending solvents comprise water.

In an embodiment of the disclosure, the ratio of the macroalgae and/ormicroalgae to the first blending solvent is 1:3 to 1:30 (w/w) of algaeto water, optionally about 1:8 to about 1:10 (w/w).

In an embodiment, the temperature suitable for phytase activity isbetween 20° C. and 60° C., optionally between 40° C. and 55° C.,suitably between 50° C. and 55° C. In another embodiment, the pHsuitable for phytase activity is between 2.0 and 7.0, optionally between4.0 and 6.0, suitably between 4.5 and 5.5, optionally 5.0 to 5.5. Inanother embodiment, the concentration of the phytase enzyme is between0.01% to 1.0% (w/w) based on the weight of the macroalgae and/ormicroalgae, optionally 0.01% and 0.5% optionally 0.01% and 0.1%. Inanother embodiment, the mixture is incubated with the phytase enzymeunder good agitation. The addition of phytase enzyme to the proteinmixture results in the hydrolysis of phytates and/or phytic acid presentin the macroalgae and/or microalgae to organic phosphates and inositol.It is known to those skilled in the art that phytates and phytic acidmay constitute undesirable anti-nutritional compounds in a proteinalgae, and accordingly, are desirably removed from the macroalgae and/ormicroalgae and the final protein products. Accordingly, the addition ofphytase enzyme results in the hydrolysis of the phytates and/or phyticacid which are subsequently removed from the mixture. In addition, ithas also been determined that phytates and/or phytic acid complex withproteins to form an insoluble gel complex. Accordingly, in anembodiment, when filtration, such as diafiltration or ultrafiltration,is utilized to purify and separate protein concentrates and/or proteinisolates, the insoluble protein/phytate (or phytic acid) gel complexesblock the filtration apparatus, reducing the flow through the filtrationapparatus, and accordingly, reducing the amount of recoverable proteinand filtration efficiency. It will be understood that the addition ofphytase to the mixture and the conditions recited for reduction orremoval of phytates and/or phytic acid apply to all of the processes andembodiments of the present disclosure.

In another embodiment of the disclosure, after treating the mixture withthe phytase enzyme, the pH of the mixture is optionally adjusted to a pHof about 6.0 to about 10.0, optionally 6.5 to about 9.5, suitably 7.0 to8.0, using a base, such as sodium hydroxide, potassium hydroxide, etc.In an embodiment, adjusting the pH of the mixture results in the proteinbecoming more soluble in the blending solvent, such as water, whichconsequently increases the yield of the protein concentrate.

In another embodiment of the disclosure, the mixture is subjected to ag-force sufficient to separate the mixture to form a fiber fraction andprotein fractions comprising an insoluble protein fraction and a solubleprotein fraction. The separation of the mixture using a sufficientg-force is described herein with reference to a centrifuge, such as adecanter centrifuge or a disc stack centrifuge, but a person skilled inthe art would understand that other methods of separation that create aseparation force, including a hydrocyclone, are also included.Accordingly, in an embodiment, when the mixture is subjected to asufficient g-force using a centrifuge, the mixture separates intothree-phases as a result of the sedimentation principle: (i) aninsoluble fiber fraction, and (ii) protein fractions comprising (ii.a)an insoluble protein fraction, and (ii.b) a soluble protein fraction.The centripetal acceleration acting on the mixture results in theinsoluble fiber fraction, which has a relatively higher density and/orgreater particle size compared to the other fractions, moving furtheralong the radial direction in which the centripetal force is acting(perpendicular to the axis of rotation). When a centrifuge is utilized,the insoluble fiber fraction (or phase) moves towards the bottom of thecentrifuge tube, as a result of its relatively higher density and/orgreater particle size, resulting in one of the phases of separation. Asa result of the proteins in the insoluble protein fraction having alower relative density and/or smaller particle size as compared to theinsoluble fiber fraction, the insoluble protein fraction forms anotherphase of separation (the middle phase). Finally, the proteins in thesoluble protein fraction, being soluble in the blending solvent and/orhaving a lower relative density compared to the fiber fraction andinsoluble protein fraction, remain near the top of the centrifuge tube.If macroalgae and/or microalgae is partially defatted algae, a fourthphase may also form on top of the soluble protein phase comprisingresidual oil. It will be understood that subjecting the mixture to asufficient g-force will not result in a total separation of the threefractions, and accordingly, a minor amount of fiber will be present inthe protein fraction, while protein will be present in the insolublefiber fraction. There will be a certain amount of protein trapped withinthe structure of the insoluble fiber fraction that is not separableusing mechanical means (i.e. using a centrifuge). In an embodiment, theamount of protein trapped within the fiber fraction will be 30%,optionally 20%, 10%, 5%, 1%. In another embodiment, proteases are usedto hydrolyze the protein trapped within the fiber fraction, whichreleases the protein from the fiber, and can be recovered therefrom, andsuch a process is also included in the present disclosure. In anotherembodiment, proteases (such as Protames, Alcalase 2.4L FG and/orFlavourzyme 1000L) are used to hydrolyze the protein trapped within thefiber fraction, which releases the protein from the fiber, and can berecovered therefrom, and such a process is also included in the presentdisclosure. In this embodiment, the hydrolyzed protein is separated fromthe fiber using any of the means disclosed herein (e.g. centrifugation,hydrocyclone). It will be understood that the disclosure concerning theg-force sufficient to separate the mixture as described above applies toall of the processes and embodiments of the present disclosure.

In an embodiment, the mixture is subjected to a g-force of between 100 gand 500 g, suitably between 150 g and 400 g, optionally between 180 gand 350 g. Calculation of g-force (or relative centrifugal force)optionally involves the RPMs (revolutions per minute) of the device, aswell as the rotational radius (in centimeters):

g-force=(RPM)²*(rotational radius)*(0.00001118)

A person skilled in the art will readily be able to calculate theg-force from the RPMs of a given separation device, such as a centrifugeor a hydrocyclone.

In another embodiment, separating the mixture comprises using acentrifuge or a hydrocyclone. In another embodiment, the centrifugecomprises a decanter centrifuge or a disc stack centrifuge.

In another embodiment, as there will be a residual amount of protein inthe separated fiber fraction, the separated fiber fraction is washedwith a second blending solvent, optionally at least once, optionallytwice or more than twice, and the mixture is then again subjected to ag-force to separate the mixture to form a fiber fraction and a proteinfraction comprising an insoluble protein fraction and a soluble proteinfraction.

In another embodiment, the separation of the insoluble fiber fractionfrom the protein fractions, results in fiber solids which are dried andconsequently constitute a high fiber algae fraction containing a lowconcentration of anti-nutritional factors, such as phytates and/orphytic acid.

In another embodiment, the pH of the protein fraction comprising theinsoluble protein fraction and soluble protein fraction is adjustedusing an acid, such as phosphoric acid, nitric acid, citric acid,sulfuric acid, and the pH is adjusted to between 4.0 and 6.0, optionally4.0 and 5.0, suitably 4.0 and 4.5. In an embodiment, adjusting the pH ofthe protein fraction using an acid results in undesirable ash becomingsoluble in the protein fraction, and therefore, separable from the finalprotein concentrate.

In another embodiment, the protein fraction comprising the insolubleprotein fraction and the soluble protein fraction is heated to atemperature between 80° C. and 100° C., optionally 90° C. and 100° C.,suitably 95° C. and 100° C., for a time period of between 5 minutes and60 minutes, optionally 5 minutes and 45 minutes, suitably between 10minutes and 30 minutes. Increasing the temperature of the proteinfractions denatures some of the undenatured proteins in the solubleprotein fraction, rendering them insoluble, and therefore increasing theyield of the insoluble protein concentrate.

In a further embodiment, separating the precipitated proteins comprisesusing a centrifuge or a hydrocyclone. In another embodiment, separatingthe precipitated proteins comprises using a centrifuge, such as decantercentrifuge or a disc stack centrifuge. In another embodiment,centrifuging the precipitated proteins comprises a g-force between 2,500g and 9,500 g. When the centrifuge is operated at such a g-force, theprecipitated proteins move along the radial axis to the bottom of thecentrifuge tube and are easily separated from the supernatant.

In another embodiment of the disclosure, the process further comprisingthe step of drying the protein concentrate to a moisture content ofbetween 4% and 8%, optionally 6% (w/w). In another embodiment, thedrying is performed using a fluidized bed dryer, conveyor dryer, rotarydryer, drum dryer, spray drier or a ring drier.

In another embodiment, the protein concentrate comprises a hydrolyzedprotein concentrate. In another embodiment, the hydrolyzed proteinconcentrate comprises peptides and/or free amino acids.

The present disclosure also includes a process for the production of aprotein concentrate from macroalgae and/or microalgae as shown in FIG.33, comprising:

i) mixing the macroalgae and/or microalgae with a first blending solventto form a mixture;

ii) optionally treating the mixture with phytase at a temperature and apH suitable for phytase activity;

iii) optionally adjusting the pH of the mixture to a pH between 6.0 and10.0;

iv) subjecting the mixture to a g-force sufficient to separate themixture to form

-   -   a) a fiber fraction, and    -   b) protein fractions comprising an insoluble protein fraction        and a soluble protein fraction;

v) optionally mixing the fiber fraction with a second blending solventand repeating step iv);

vi) optionally adjusting the pH of the protein fraction to a pH between4.0 and 6.0;

vii) mixing the protein fraction with a mixing solvent to form a proteinslurry and precipitate the proteins;

viii) separating the precipitated proteins from the protein slurry toform the protein concentrate; and

ix) optionally repeating steps vii) and viii) with the precipitatedproteins.

In another embodiment, the first and second blending solvents comprisewater, a saline solution or a polysaccharide solution. In a furtherembodiment, the first and second blending solvents comprise water.

In an embodiment of the disclosure, the ratio of the macroalgae and/ormicroalgae to the first blending solvent is 1:3 to 1:30 (w/w) of algaeto water, optionally about 1:8 to about 1:10 (w/w).

In an embodiment, the temperature suitable for phytase activity isbetween 20° C. and 60° C., optionally between 40° C. and 55° C.,suitably between 50° C. and 55° C. In another embodiment, the pHsuitable for phytase activity is between 2.0 and 7.0, optionally between4.0 and 6.0, suitably between 4.5 and 5.5, optionally 5.0 to 5.5. Inanother embodiment, the concentration of the phytase enzyme is between0.01% to 1.0% (w/w) based on the weight of the macroalgae and/ormicroalgae, optionally 0.01% and 0.5% optionally 0.01% and 0.1%. Inanother embodiment, the mixture is incubated with the phytase enzymeunder good agitation.

In another embodiment of the disclosure, after treating the mixture withthe phytase enzyme, the pH of the mixture is optionally adjusted to a pHof about 6.0 to about 10.0, optionally 6.5 to about 9.5, suitably 7.0 to8.0, using a base, such as sodium hydroxide, potassium hydroxide, etc.In an embodiment, adjusting the pH of the mixture results in the proteinbecoming more soluble in the blending solvent, such as water, whichconsequently increases the yield of the protein concentrate.

In another embodiment of the disclosure, the mixture is subjected to ag-force of between 100 g and 500 g, suitably between 150 g and 400 g,optionally between 180 g and 350 g.

In another embodiment, separating the mixture comprises using acentrifuge or a hydrocyclone. In another embodiment, the centrifugecomprises a decanter centrifuge or disc stack centrifuge.

In another embodiment, as there will be a residual amount of protein inthe separated fiber fraction, the separated fiber fraction is washedwith a second blending solvent, optionally at least once, optionallytwice or more than twice, and the mixture is then again subjected to ag-force to separate the mixture to form a fiber fraction and a proteinfraction comprising an insoluble protein fraction and a soluble proteinfraction.

In another embodiment, the separation of the insoluble fiber fractionfrom the protein fraction, results in fiber solids which are dried andconsequently constitute a high fiber algae fraction containing a lowconcentration of anti-nutritional factors, such as phytates and/orphytic acid.

In another embodiment, the pH of the protein fraction comprising theinsoluble protein fraction and soluble protein fraction is adjustedusing an acid, such as phosphoric acid, nitric acid, citric acid,sulfuric acid, hydrochloric acid, and the pH is adjusted to between 4.0and 6.0, optionally 4.0 and 5.0, suitably 4.0 and 4.5. In an embodiment,adjusting the pH of the protein fraction using an acid results inundesirable ash becoming soluble in the protein fraction, and therefore,separable from the final protein concentrate.

In another embodiment of the disclosure, the protein fraction is mixedwith a mixing solvent comprising an ethanol:water mixture, wherein theethanol is present in an amount between 90% and 100%, optionally 95% and100% (v/v). It will be understood that 100% ethanol may contain a smallpercentage of impurities such as water, etc., which cannot be removedfrom the ethanol. In an embodiment, mixing solvent is added to theprotein fraction at a ratio of between 2:1 and 1:2 (v/v of mixingsolvent:protein fraction), optionally 1:1. In an embodiment, when themixing solvent comprises an alcohol, such as ethanol (80%, 90%, 95%ethanol in water or 100% ethanol), proteins in the protein slurryprecipitate from solution, as a result the proteins being less solublein the mixing solvent (such as ethanol) than in the blending solvent(such as water), and therefore increases the yield of the proteinconcentrate.

In another embodiment, separating the precipitated proteins comprisesusing a centrifuge or a hydrocyclone. In another embodiment, separatingthe precipitated proteins comprises using a centrifuge, such as decantercentrifuge or disc stack centrifuge. In another embodiment, centrifugingthe precipitated proteins comprises a g-force between 2,500 g and 9,500g. When the centrifuge is operated at such a g-force, the precipitatedproteins move along the radial axis to the bottom of the centrifuge tubeand are easily separated from the supernatant.

In another embodiment of the disclosure, steps viii) and viii) arerepeated at least twice, such that the precipitated proteins are washedwith mixing solvent to remove impurities.

In another embodiment, the process further comprises the step of dryingthe protein concentrate to a moisture content of between 4% and 8%(w/w), optionally 6% (w/w). In another embodiment, the drying isperformed using a fluidized bed dryer, spray dryer or a ring drier.

In another embodiment, the protein concentrate comprises a hydrolyzedprotein concentrate. In a further embodiment, the hydrolyzed proteinconcentrate comprises peptides and/or free amino acids.

The present disclosure also includes a process for the production of aprotein isolate from macroalgae and/or microalgae as shown in FIG. 34,comprising:

i) mixing the macroalgae and/or microalgae with a first blending solventto form a mixture;

ii) optionally treating the mixture with phytase at a temperature and apH suitable for phytase activity;

iii) optionally adjusting the pH of the mixture to a pH between 6.0 and10.0;

iv) subjecting the mixture to a g-force sufficient to separate themixture to form

-   -   a) a fiber fraction, and    -   b) protein fractions comprising an insoluble protein fraction        and a soluble protein fraction;

v) optionally mixing the fiber fraction with a second blending solventand repeating step iv);

vi) separating the insoluble protein fraction from the soluble proteinfraction to recover therefrom an insoluble protein concentrate and asoluble protein extract; and

vii) subjecting the soluble protein extract to filtration to recovertherefrom the protein isolate.

In another embodiment, the first and second blending solvents comprisewater, a saline solution or a polysaccharide solution. In a furtherembodiment, the first and second blending solvents comprise water.

In an embodiment of the disclosure, the ratio of the macroalgae and/ormicroalgae to the first blending solvent is 1:3 to 1:30 (w/w) of algaeto water, optionally 1:8 to 1:10 (w/w).

In an embodiment, the temperature suitable for phytase activity isbetween 20° C. and 60° C., optionally between 40° C. and 55° C.,suitably between 50° C. and 55° C. In another embodiment, the pHsuitable for phytase activity is between 2.0 and 7.0, optionally between4.0 and 6.0, suitably between 4.5 and 5.5, optionally 5.0 and 5.5. Inanother embodiment, the concentration of the phytase enzyme is between0.01% and 1.0% (w/w) based on the weight of the macroalgae and/ormicroalgae, optionally 0.01% and 0.5% optionally 0.01% and 0.1%. Inanother embodiment, the mixture is incubated with the phytase enzymeunder good agitation.

In another embodiment of the disclosure, after treating the mixture withthe phytase enzyme, the pH of the mixture is optionally adjusted to a pHof between 6.0 and about 10.0, optionally 7.0 and 9.0, suitably 7.0 and8.0, using a base, such as sodium hydroxide, potassium hydroxide, etc.In an embodiment, adjusting the pH of the mixture results in the proteinbecoming more soluble in the blending solvent, such as water, whichconsequently increases the yield of the protein isolate.

In another embodiment of the disclosure, the mixture is subjected to ag-force of between 100 g and 500 g, suitably between 150 g and 400 g,optionally between 180 g and 350 g.

In another embodiment, separating the mixture comprises using acentrifuge or a hydrocyclone. In an embodiment, the centrifuge comprisesa decanter centrifuge or disc stack centrifuge.

In another embodiment, as there will be a residual amount of protein inthe separated fiber fraction, the separated fiber fraction is washedwith a second blending solvent, optionally at least once, optionallytwice or more than twice, and the mixture is then again subjected to ag-force to separate the mixture to form a fiber fraction and a proteinfraction comprising an insoluble protein fraction and a soluble proteinfraction.

In another embodiment, the separation of the insoluble fiber fractionfrom the protein fraction, results in fiber solids which are dried andconsequently constitute a high fiber algae fraction containing a lowconcentration of anti-nutritional factors, such as phytates and/orphytic acid.

In another embodiment, separating the insoluble protein fraction fromthe soluble fiber fraction comprises using a centrifuge or ahydrocyclone. In a further embodiment separating the insoluble proteinfraction from the soluble protein fraction comprises using a centrifuge,such as a decanter centrifuge or disc stack centrifuge.

In another embodiment, centrifuging to separate the insoluble proteinfraction from the soluble protein fraction comprises a g-force between2,500 g and 9,500 g. In another embodiment, the separation of theinsoluble protein fraction from the soluble protein fraction results ina wet protein concentrate that can be subsequently dried. The extractfrom the separation of the insoluble protein fraction from the solubleprotein fraction comprises the soluble protein, which is subsequentlyfiltered through a filtration apparatus, such as ultrafiltration and/ordiafiltration, resulting in the protein isolate. As described above,phytates and/or phytic acid can complex and bind to the proteins, andconsequently block the filtration apparatus. The removal of the phytatesand/or phytic acid from the macroalgae and/or microalgae mixture (algaeand blending solvent) using phytase as described above, such that thefiltration apparatus is not blocked with such complexes, resulting thefiltration apparatus performing efficiently to produce the proteinisolate.

In another embodiment, the process further comprises the step of dryingthe protein isolate to a moisture content of between 4% and 8% (w/w),optionally 6% (w/w). In another embodiment, the drying is performedusing a spray drier or a ring drier.

In a further embodiment, the protein isolate comprises a hydrolyzedprotein isolate. In another embodiment, the hydrolyzed proteinconcentrate comprises peptides and/or free amino acids.

In another embodiment, the present disclosure includes a process forobtaining a protein concentrate from macroalgae and/or microalgaecomprising:

i) mixing the macroalgae and/or microalgae with a first blending solventto form a mixture;

ii) optionally treating the mixture with phytase at a temperature and apH suitable for phytase activity;

iii) optionally adjusting the pH of the mixture to a pH suitable tosolubilize proteins in the mixture;

iv) subjecting the mixture to a g-force sufficient to separate themixture to form

-   -   a) a fiber fraction, and    -   b) a protein fraction comprising        -   (i) an insoluble protein fraction, and        -   (ii) a soluble protein fraction;

v) separating the fiber fraction from the protein fraction and mixingthe fiber fraction with a second blending solvent to form a fibermixture;

vi) treating the fiber mixture with a protease at a temperature and a pHsuitable for protease activity;

vii) subjecting the fiber mixture to a g-force sufficient to separatethe fiber mixture to form:

-   -   a) a second fiber fraction, and    -   b) a hydrolyzed protein fraction, comprising        -   (i) an insoluble protein fraction comprising partially            hydrolyzed and un-hydrolyzed protein, and        -   (ii) a soluble hydrolyzed protein fraction;

viii) optionally adjusting the pH of the protein fraction from stepiv(b) to a pH suitable to precipitate the proteins;

x) separating the precipitated proteins from the protein fraction;

xi) optionally combining the precipitated proteins and the hydrolyzedprotein fraction to form the protein concentrate.

In one embodiment, the pH of the mixture (algae and first blendingsolvent) is optionally adjusted to solubilize proteins (undenaturedproteins) into the first blending solvent. In an embodiment, the pH ofthe mixture is adjusted to a pH between 6 and 8, optionally between 6.5and 7.5, optionally about 7.0, using a base such as sodium hydroxide,which solubilizes some of the proteins present in the algae, and uponlow g-force separation, increases the amount of protein in the proteinfraction. In addition, adjusting the pH to about 7.0 results in thefiber fraction (after low g-force separation of the mixture) having aneutral pH as a by-product.

In another embodiment, the process further comprises mixing the fiberfraction with the first blending solvent and repeating step iv) once,twice or three times and/or mixing the second fiber fraction with thesecond blending solvent and repeating step vii) once, twice or threetimes. The repeated washing of the fiber fractions with the blendingsolvent increases the amount of protein that is recovered for theprotein concentrate as the repeated washings separates additionalprotein that is trapped within the fiber structure.

In another embodiment, the first and second blending solvents comprisewater, a saline solution or a polysaccharide solution, optionally water,and wherein the ratio of the macroalgae and/or microalgae to the firstblending solvent is 1:3 to 1:30 (w/w) of algae to water, optionally 1:8.

In another embodiment, the temperature suitable for phytase activity isbetween 20° and 60° C. and the pH suitable for phytase activity isbetween 2.0 and 7.0 and the temperature suitable for protease activityis between 30° and 70° C. and the pH suitable for protease activity isbetween 5.0 and 9.0.

In another embodiment, the mixture and/or the fiber mixture is subjectedto a g-force of between 100 g and 500 g, optionally between 150 g and400 g, or between 170 g and 350 g. In an embodiment, separating themixture and/or the fiber mixture comprises using a centrifuge or ahydrocyclone, optionally a decanter centrifuge.

In a further embodiment, the pH of the protein fraction from step iv(b)is adjusted to precipitate proteins in the protein fraction. The proteinfraction comprises (i) an insoluble protein fraction, and (ii) a solubleprotein fraction, and the adjustment of the pH precipitates proteinsfrom the soluble protein fraction, and therefore increases the yield ofthe protein concentrate. In one embodiment, the pH of the proteinfraction is adjusted to the isoelectric point of the soluble protein, atwhich point the soluble protein precipitates from solution. In oneembodiment, the pH suitable to precipitate the proteins in the proteinfraction is between 4.0 and 6.0, optionally 4.0 and 5.0, suitably 4.0and 4.5 using an acid such as phosphoric acid, nitric acid, sulfuricacid, hydrochloric acid, optionally phosphoric acid having aconcentration of 40% to 60% (w/w), optionally about 50%. In anotherembodiment, adjusting the pH of the protein fraction using an acidresults in undesirable ash becoming soluble in the protein fraction, andtherefore, separable from the final protein concentrate.

In another embodiment, separating the precipitated proteins comprisesusing a centrifuge or a hydrocyclone. In another embodiment, separatingthe precipitated proteins comprises using a centrifuge, such as decantercentrifuge or disc stack centrifuge. In another embodiment, centrifugingthe precipitated proteins comprises a g-force between 2,500 g and 9,500g. When the centrifuge is operated at such a g-force, the precipitatedproteins move along the radial axis to the bottom of the centrifuge tubeand are easily separated from the supernatant.

In another embodiment, the process further comprises the step of dryingthe protein concentrate to a moisture of between 4% and 8% (w/w). Inanother embodiment, the protein concentrate also comprises peptides andfree amino acids.

The present disclosure also includes a process for the production of aprotein concentrate from macroalgae and/or microalgae comprising:

i) mixing the macroalgae and/or microalgae with a first blending solventto form a mixture;

ii) optionally treating the mixture with phytase at a temperature and apH suitable for phytase activity;

iii) optionally adjusting the pH of the mixture to a pH suitable tosolubilize proteins in the mixture;

iv) subjecting the mixture to a g-force sufficient to separate themixture to form

-   -   a) a fiber fraction, and    -   b) a protein fraction comprising        -   (i) an insoluble protein fraction, and        -   (ii) a soluble protein fraction;

v) separating the fiber fraction from the protein fraction and mixingthe fiber fraction with a second blending solvent to form a fibermixture;

vi) treating the fiber mixture with a protease at a temperature and a pHsuitable for protease activity;

vii) subjecting the fiber mixture to a g-force sufficient to separatethe fiber mixture to form:

-   -   a) a second fiber fraction, and    -   b) a hydrolyzed protein fraction, comprising        -   (i) an insoluble protein fraction, and        -   (ii) a soluble hydrolyzed protein fraction;

ix) mixing the protein fraction with a mixing solvent to precipitateproteins;

x) separating the precipitated proteins from the protein fraction; and

xi) optionally combining the precipitated proteins and the hydrolyzedprotein fraction to form the protein concentrate.

In one embodiment, the pH of the mixture (algae and first blendingsolvent) is optionally adjusted to solubilize proteins (undenaturedproteins) into the first blending solvent. In an embodiment, the pH ofthe mixture is adjusted to a pH between 6 and 8, optionally between 6.5and 7.5, optionally about 7.0, using a base such as sodium hydroxide,which solubilizes some of the proteins present in the algae, and uponlow g-force separation, increases the amount of protein in the proteinfraction. In addition, adjusting the pH to about 7.0 results in thefiber fraction (after low g-force separation of the mixture) having aneutral pH as a by-product.

In another embodiment, the process further comprises mixing the fiberfraction with the first blending solvent and repeating step iv) once,twice or three times and/or mixing the second fiber fraction with thesecond blending solvent and repeating step vii) once, twice or threetimes. The repeated washing of the fiber fractions with the blendingsolvent increases the amount of protein that is recovered for theprotein concentrate as the repeated washings separates additionalprotein that is trapped within the fiber structure.

In another embodiment, the first and second blending solvents comprisewater, a saline solution or a polysaccharide solution, optionally water,and wherein the ratio of the macroalgae and/or microalgae to the firstblending solvent is 1:3 to 1:30 (w/w) of algae to water, optionally 1:8.

In another embodiment, the temperature suitable for phytase activity isbetween 20° and 60° C. and the pH suitable for phytase activity isbetween 2.0 and 7.0 and the temperature suitable for protease activityis between 30° and 70° C. and the pH suitable for protease activity isbetween 5.0 and 9.0.

In another embodiment, the mixture and/or the fiber mixture is subjectedto a g-force of between 100 g and 500 g, optionally between 150 g and400 g, or between 170 g and 350 g. In an embodiment, separating themixture and/or the fiber mixture comprises using a centrifuge or ahydrocyclone, optionally a decanter centrifuge.

In another embodiment, the pH of the protein fraction from step iv(b) isadjusted using an acid, such as phosphoric acid, nitric acid, citricacid, sulfuric acid, hydrochloric acid, and the pH is adjusted tobetween 4.0 and 6.0, optionally 4.0 and 5.0, suitably 4.0 and 4.5. In anembodiment, adjusting the pH of the protein fraction using an acidresults in undesirable ash becoming soluble in the protein fraction, andtherefore, separable from the final protein concentrate.

In another embodiment of the disclosure, the protein fraction is mixedwith a mixing solvent comprising an ethanol:water mixture, wherein theethanol is present in an amount between 80% and 100%, optionally 90% and100%, optionally 95% and 100% (v/v). It will be understood that 100%ethanol may contain a small percentage of impurities such as water,etc., which cannot be removed from the ethanol. In an embodiment, mixingsolvent is added to the protein fraction at a ratio of between 2:1 and1:2 (v/v of mixing solvent:protein fraction), optionally 1:1. In anembodiment, when the mixing solvent comprises an alcohol, such asethanol (80%, 90%, 95% ethanol in water or 100% ethanol), solubleproteins in the protein fraction precipitate from solution, as a resultthe proteins being less soluble in the mixing solvent (such as ethanol)than in the blending solvent (such as water), and therefore increasesthe yield of the protein concentrate.

In another embodiment, the mixing solvent comprises an ethanol:watermixture, wherein the ethanol is present in an amount between 80% and100% (v/v). The addition of the mixing solvent to the protein fractionfrom step iv(b) causes proteins in the protein fraction to precipitateas a result of the proteins having a lower solubility in such a solvent(such as ethanol/water mixture). Accordingly, the amount of proteinrecovered is increased upon separation.

In another embodiment, separating the precipitated proteins comprisesusing a centrifuge or a hydrocyclone. In another embodiment, separatingthe precipitated proteins comprises using a centrifuge, such as decantercentrifuge or disc stack centrifuge. In another embodiment, centrifugingthe precipitated proteins comprises a g-force between 2,500 g and 9,500g. When the centrifuge is operated at such a g-force, the precipitatedproteins move along the radial axis to the bottom of the centrifuge tubeand are easily separated from the supernatant.

In another embodiment, the process further comprises the step of dryingthe protein concentrate to a moisture of between 4% and 8% (w/w). Inanother embodiment, the protein concentrate also comprises peptides andfree amino acids.

In another embodiment, the present disclosure also includes a processfor the production of a protein isolate from macroalgae and/ormicroalgae comprising:

i) mixing the macroalgae and/or microalgae with a first blending solventto form a mixture;

ii) optionally treating the mixture with phytase at a temperature and apH suitable for phytase activity;

iii) optionally adjusting the pH of the mixture to a pH suitable tosolubilize proteins;

iv) subjecting the mixture to a g-force sufficient to separate themixture to form

-   -   a) a fiber fraction, and    -   b) a protein fraction comprising        -   (i) an insoluble protein fraction, and        -   (ii) a soluble protein fraction;

vi) separating the insoluble protein fraction from the soluble proteinfraction to recover therefrom an insoluble protein concentrate and asoluble protein extract; and

vii) subjecting the soluble protein extract to filtration to recovertherefrom the protein isolate.

In one embodiment, the pH of the mixture (algae and first blendingsolvent) is optionally adjusted to solubilize proteins (undenaturedproteins) into the first blending solvent. In an embodiment, the pH ofthe mixture is adjusted to a pH between 6 and 8, optionally between 6.5and 7.5, optionally about 7.0, using a base such as sodium hydroxide,which solubilizes some of the proteins present in the macroalgae and/ormicroalgae, and upon low g-force separation, increases the amount ofprotein in the protein fraction. In addition, adjusting the pH to about7.0 results in the fiber fraction (after low g-force separation of themixture) having a neutral pH as a by-product.

In another embodiment, the process further comprises mixing the fiberfraction with the first blending solvent and repeating step iv) once,twice or three times. The repeated washing of the fiber fraction withthe blending solvent increases the amount of protein that is recoveredfor the protein concentrate as the repeated washings separatesadditional protein that is trapped within the fiber structure.

In another embodiment, the first and second blending solvents comprisewater, a saline solution or a polysaccharide solution, optionally water,and wherein the ratio of the macroalgae and/or microalgae to the firstblending solvent is 1:3 to 1:30 (w/w) of algae to water.

In another embodiment, the temperature suitable for phytase activity isbetween 20° and 60° C. and the pH suitable for phytase activity isbetween 2.0 and 7.0 and the temperature suitable for protease activityis between 30° and 70° C. and the pH suitable for protease activity isbetween 5.0 and 9.0.

In another embodiment, the mixture and/or the fiber mixture is subjectedto a g-force of between 100 g and 500 g, optionally between 150 g and400 g, or between 170 g and 350 g. In an embodiment, separating themixture and/or the fiber mixture comprises using a centrifuge or ahydrocyclone, optionally in a decanter centrifuge.

In another embodiment, centrifuging to separate the insoluble proteinfraction from the soluble protein fraction comprises a g-force between2,500 g and 9,500 g. In another embodiment, the separation of theinsoluble protein fraction from the soluble protein fraction results ina wet protein concentrate that can be subsequently dried. The extractfrom the separation of the insoluble protein fraction from the solubleprotein fraction comprises the soluble protein, which is subsequentlyfiltered through a filtration apparatus, such as ultrafiltration and/ordiafiltration, resulting in the protein isolate. As described above,phytates and/or phytic acid can complex and bind to the proteins, andconsequently block the filtration apparatus. The removal of the phytatesand/or phytic acid from the macroalgae and/or microalgae mixture(macroalgae and/or microalgae and blending solvent) using phytase asdescribed above, such that the filtration apparatus is not blocked withsuch complexes, resulting the filtration apparatus performingefficiently to produce the protein isolate.

In another embodiment, the process further comprises the step of dryingthe protein concentrate to a moisture of between 4% and 8% (w/w). Inanother embodiment, the protein concentrate also comprises peptides andfree amino acids.

In another embodiment, any of the above processes is conducted using acounter-current process.

In another embodiment, there is also included a process for treatingmacroalgae and/or microalgae comprising a fiber fraction and a proteinfraction, wherein the protein fraction comprises (i) an insolubleprotein fraction and (ii) a soluble protein fraction, to separate thefiber fraction from the protein fraction comprising:

i) mixing the macroalgae and/or microalgae with a first blending solventto form a mixture;

ii) optionally adjusting the pH of the mixture to a pH suitable tosolubilize proteins in the mixture;

iv) subjecting the mixture to a g-force sufficient to separate themixture to form

-   -   a) a fiber fraction, and    -   b) a protein fraction comprising        -   (i) an insoluble protein fraction, and        -   (ii) a soluble protein fraction; and

v) separating the fiber fraction from the protein fraction.

In one embodiment, the separation at a g-force of between 150 to 300 g,optionally 150 to 200 g, optionally 175 to 180 g, comprises using anymeans to achieve such g-forces, such as a centrifuge, such as a decantercentrifuge, or a hydrocylcone.

Terms of degree such as “substantially”, “about” and “approximately” asused herein mean a reasonable amount of deviation of the modified termsuch that the end result is not significantly changed. These terms ofdegree should be construed as including a deviation of at least ±5% ofthe modified term if this deviation would not negate the meaning of theword it modifies.

The following non-limiting examples are illustrative of embodiments ofthe present disclosure:

EXAMPLES Reagents and Materials

Commercial methyl pentane was purchased from Univar Canada Ltd.,Saskatoon, Saskatchewan, Canada. Enzyme samples of Cellulase(Celluclast® 1.5 L), Cellulase Complex, Alcalase® 2.4L FG, andFlavourzyme® were obtained from Novozymes North America, Inc.,Franklinton, N.C. USA.

Defatted microalgae were produced from dried microalgae (Chlorella)through the milling of microalgae in methyl pentane using a bead mill,solids-liquid separation by centrifugation using a decanter,desolventization and drying using a down draft desolventizer. Defattedmacroalgae were produced from dried macroalgae (Kelp) through themilling of microalgae in methyl pentane using a bead mill, solids-liquidseparation by centrifugation using a decanter, desolventization anddrying using a down draft desolventizer.

Alcalase 2.4L FG was obtained from Novozymes North America, Inc.,Franklinton, N.C. USA. Alcalase 2.4L FG is a proteolytic enzyme producedby submerged fermentation of a selected strain of Bacilluslicheniformis.

Two samples of phytase (Natuphos. powder form, 10,000 FTU/g) and phytase(Natuphos. powder form, 55,000 FTU/g) were purchased from BASF Canada,Georgetown, Ontario, Canada.

Analysis

Mixing of the materials was performed using a Ribbon Blender (TorcoModel R-12, Toronto Coppersmithing International Ltd., Scarborough,Ontario, Canada). Heat treatments of samples were conducted using anInfra Red Cereal Processing System (Micronizing Company Limited,Framlingham, Sulfolk, England) or a two-tray Simon-Rosedown cooker(Laboratory cooker-press, Simon-Rosedowns Limited, Hull, England).Ultrafiltration was carried out using a Millipore® Ultrafiltration Unit(Model A60, Millipore® Corporation, Bedford, Mass., USA). Proteincontent of the samples was determined by the Leco® Protein Analyzer(Model FP-428, Leco® Corporation, ST. Joseph, Mich. U.S.A.) based onAOCS Official Method Ba 4e-93. Moisture content of the samples wasdetermined by drying samples in a 105±2° C. convection oven for 16 hoursor to a constant weight based on AOCS Official Method Ba 2a-38. Oilcontent of the samples was determined based on AOCS Official Method Ba3-38 with the following changes: (a) 2 g of sample was used instead of 5g in the analysis; (b) extraction continued for 4 hours, and (c)extraction flask was heated to remove residual petroleum ethers. Ashcontent of the samples was determined based on AOCS Official Method Ba5a-49 with the following changes: (a) samples were pre-ashed on a hotplate prior to being placed into the muffle furnace; (b) samples wereincinerated for 18 hours in muffle furnace; and (c) nitric acid wasadded if sample remained black. Crude fiber content of the samples wasdetermined based on AOCS Official Method Ba 6-84 with the followingchanges: (a) samples with oil contents below 3% were not defatted and(b) digest was dried for 2 hours at 130° C. Protein dispersibility index(PDI) of the samples was determined based on A.O.C.S. Official Method Ba10-65. Free fatty acid (FFA) of the oil samples was determined based onAOCS Official Method Ca 5a-40. Phosphorus and sulphur of the sampleswere determined based on the modified methods of AOCS Ca20-99 and AOCSCa 17-01 (modified), respectively. Crude fiber content of the sampleswas determined based on AOCS Official Method Ba 6-84 with the followingchanges: (a) samples with oil contents below 3% were not defatted and(b) digest was dried for 2 hours at 130° C. Protein dispersibility index(PDI) of the samples was determined based on A.O.C.S. Official Method Ba10-65. Glucosinolate content of the samples was determined based on theMethod of the Canadian Grain Commission, Grain Research Laboratory(Daun, J. K. and McGregor, D. I., Glucosinolate Analysis of Rapeseed(Canola), Dec. 15, 1981). Solvent residues were determined using GC/MStechniques based on a modified method of A.O.C.S. Official Method, Ba13-87.

Example 1 Prophetic Preparation of Protein Concentrate Using Phytasefrom Algae

Approximately 15 kg dried microalgae or macroalgae is mixed with 120 kgof tab water at ambient temperature. Approximately 12 g of phytase(Natuphos 10,000 L Phytase) at 0.08% dosage based on the starting weightof dried microalgae or macroalgae is added to the slurry. The pH ofalgae slurry is adjusted to 5.5 and temperature to 50° C. After holdingfor 1.5 hours under agitation, the slurry is centrifuged at 180 g force(1500 RPM bowl speed) to separate fiber solids from algae protein slurry(slurry 1) containing soluble and insoluble proteins using a BirdDecanter (Bird 6″ Continuous Bowl Decanter, Saskatoon, Canada). Fibersolids is mixed with water at a ratio of 1 to 1 by weight at ambienttemperature, which is followed by centrifugation at 180 g force (1500RPM bowl speed) to separate the 1^(st) washed fiber solids from theprotein slurry (slurry 2) containing soluble and insoluble proteinsusing the Bird Decanter. The 1^(st) washed fiber solids are mixed withwater at a ratio of 1 to 1 by weight at ambient temperature. This isfollowed by centrifugation at 180 g force (1500 RPM bowl speed) toseparate the second washed fiber solids from the protein slurry (slurry3) containing soluble and insoluble proteins using the Bird Decanter.

Algae protein slurries containing soluble and insoluble proteins arecombined together. The protein slurry is mixed with 100% denaturedethanol (SDAG 13) at a volume ratio of 1 to 1 at ambient temperatureunder agitation. This is followed by centrifugation at 6,550 g force(8500 RPM) to recover precipitated proteins using a Westfalia Disc StackCentrifuge (SA-7, Oelde, Germany). The precipitated proteins are washedwith 85% ethanol (v/v) twice and centrifuged to recover the washedprotein solids using a Basket Centrifuge (Tolhurst—26″ Center-Slung,Ametek Inc., East Moline, Ill., USA). The recovered protein solids aredesolventized in a fume hood before the final drying in a vacuum traydryer to 5.59% moisture. The produced algae protein concentrate wouldcontain above 50% protein, low fiber, and low anti-nutritional factors.

Example 2 Prophetic Preparation of Protein Concentrate Using Phytasefrom Algae

Approximately 40 kg of dried microalgae or macroalgae is mixed with 320kg of tab water at ambient temperature. Approximately 32 g of phytase(Natuphos 10,000 L Phytase) at 0.08% dosage based on the starting weightof algae is added to the algae slurry. The pH of algae slurry isadjusted to 5.5 and temperature to 50° C. After holding for 1.5 hoursunder agitation, the algae slurry is centrifuged at 180 g force (1500RPM bowl speed) to separate fiber solids from algae protein slurry(slurry 1) containing soluble and insoluble proteins using the BirdDecanter (Bird 6″ Continuous Bowl Decanter, Saskatoon, Canada). Fibersolids are mixed with water at a ratio of 1 to 1 by weight at ambienttemperature, which is followed by centrifugation at 180 g force (1500RPM bowl speed) to separate the washed fiber solids from the algaeprotein slurry (slurry 2) containing soluble and insoluble proteinsusing the Bird Decanter.

Algae protein slurries containing soluble and insoluble proteins arecombined together. The pH of combined protein slurry is adjusted to4.5±0.1 by addition of 85% phosphoric acid. The protein slurry is heatedto 95-100° C. for 10 to 60 minutes. The protein slurry is cooled down to65° C. This is followed by centrifugation at 3,300 g force (5200 RPMbowl speed) to recover the majority of the precipitated proteins using aWestfalia Decanter Centrifuge (CA 225-010, Germany). The supernatantwould still contain a small amount of fine precipitated proteins thatwould be recovered by centrifugation at 6,715 g force (7560 RPM) using aWestfalia Disc Stack Centrifuge (SA-14, Germany). The precipitatedproteins are combined together, which is followed by mixing with waterat a ratio of 1 to 3 by weight, at pH4.5 and at ambient temperature. Thewashed protein precipitates are recovered by centrifugation at 3,300 gusing the Decanter at first, which is followed by centrifugation at6,715 g using the Disc Stack Centrifuge to recover the remainingprecipitated proteins. Finally, the washed protein precipitates aremixed with water at a ratio of 1 to 3 by weight, pH 4.5 and ambienttemperature. The final washed protein precipitates are recovered bycentrifugation using both the Decanter at 3,300 g and the Disc StackCentrifuge at 6,715 g.

The final washed protein precipitates are dried to about 6% moisture.The algae protein concentrate would contain over 50% protein, low fiber,low ash and low anti-nutritional factors.

Example 3 Preparation of Protein Concentrate from Microalage (i) WithoutProtein Hydrolysis—Control

Approximately 0.5 kg of defatted microalgae (Chlorella) was mixed with0.5 kg of water at ambient temperature. pH of the microalgae slurry wasat 5.76. The slurry was heated to 50° C. Approximately 0.075 g ofphytase (Natuphos, 55,000 FTU/g) was added to the slurry. The slurry washeld at 50° C. for 1 hour under agitation. pH of the slurry was adjustedto 7.0 by addition of 10% NaOH solution. This was followed bycentrifugation at 4,000 rpm for 10 minutes to separate the solubleextract from the insoluble solids using a lab centrifuge. The insolublesolids were washed with water twice. For each wash, the insoluble solidswere mixed with water at a ratio of 1 to 2 by weight, which was followedby centrifugation at 4,000 rpm for 10 minutes to separate the washedinsoluble solids from the washing extract. The washed solids were freezedried into protein concentrate. The soluble extract was combined withthe wash extracts. The combined liquid extract was subjected tountrafiltration and diafiltration in order to recover the solubleproteins and remove the ash.

(ii) Protein Hydrolysis with 0.5% Alcalase

FIGS. 35 and 36 illustrate the preparation of a protein concentrate froma toasted meal using a protease. Approximately 100 g of defattedmicroalgae meal (Chlorella) was mixed with 800 g of water at ambienttemperature. The microalgae slurry had a pH 10, which was adjusted to5.9±0.2 by addition of 50% phosphoric acid. The microalgae slurry washeated to 52±2° C. and 0.3 g of phytase (Natuphos. powder form, 10,000FTU/g) was added to the slurry. Hydrolysis of phytates was carried outat 52±2° C. for 1 hour. After hydrolysis of phytates, the pH ofmicroalgae slurry was adjusted to 7.0±0.1 by slow addition of 10% NaOHsolution.

After pH adjustment to 7.0±0.1, the microalgae slurry was centrifuged at2,000 rpm for 10 minutes using a lab centrifuge. The soluble liquidextract was separated from the insoluble solids. The insoluble solidswere mixed with water at a ratio of 1 to 1 by weight at ambienttemperature, which was followed by centrifugation at 4,000 rpm for 10minutes to separate the washed solids from the wash extract. The washedsolids were washed with water three more times. For each wash, theinsoluble solids were mixed with water at a ratio of 1 to 1 by weight.This was followed by centrifugation at 4,000 rpm to separate the washedsolids from the wash extract. The soluble extract was combined with thewashed extracts. The combined soluble extract was adjusted to lower pHby addition of 50% phosphoric acid to precipitate some proteins. Theliquid extract was separated from precipitated proteins bycentrifugation at 4,000 rpm for 10 minutes. The soluble liquid extractwas subjected to ultrafiltration and diafiltration to recover solubleproteins and to remove ash.

The final washed solids were mixed with water at a ratio of 1 to 1 byweight. pH of the slurry was adjusted to 8.3±0.1 by addition of 10% NaOHsolution. The slurry was heated to 60±2° C. 0.5 g of Alcalase (0.5%dosage based on the weight of defatted microalgae meal) was added to theslurry and protein hydrolysis was carried out at 60±2° C. for 2 hours.After protein hydrolysis, the slurry was centrifuged at 4,000 rpm for 10minutes using the lab centrifuge. The hydrolyzed protein extract wasseparated from the insoluble solids by centrifugation at 4,000 rpm for10 minutes using the lab centrifuge. The insoluble solids were mixedwith water at a ratio of 1 to 1 by weight. This was followed bycentrifugation at 4,000 rpm for 10 minutes using the lab centrifuge. Thewashed solids were again separated from the hydrolyzed protein extract.

The hydrolyzed protein extract was adjusted to pH7.0 before drying toproduce the dried protein concentrate.

The washed solids after protein hydrolysis were also dried to producethe by-product of microalgae solids.

Discussion

The results of proximate analysis of defatted microalgae, proteinconcentrate and hydrolyzed protein concentrate are shown in Table 1.Defatted microalgae meal contains 54.8% protein and 0.46% phytates on adry weight basis. The protein solubility index (PDI) is about 29.3%.

Hydrolyzed protein concentrate generated with the use of protease oneanother microalgae sample is show in Table 2. Hydrolyzed proteinconcentrate has a protein content of 58.88% (dwb). The remaininginsoluble solids after protein hydrolysis and separation have a proteincontent of 13.4% (dwb). The process of protein hydrolysis with the useof protease was effective to solubilize the previous insoluble proteins.The process was also effective to reduce the ash and fiber contents. Theash content was reduced from 35.24% in the starting microalgae to 12.15%in the hydrolyzed protein concentrate. The fiber content was reducedfrom 5.96% in the starting microalgae meal to 2.94% in the hydrolyzedprotein concentrate. The microalgae fiber solids have very high ashcontent of 41.24% and a high fiber content of 13.21% on a dry weightbasis.

As shown in FIGS. 37 and 38, the processes of the present disclosureare, in one embodiment, performed using a concurrent process (FIG. 37),or in another embodiment, a counter-current process (FIG. 38). In thecounter-current process, there is substantial savings of water andenergy as well as much better production throughput because the volumeof the process streams is greatly reduced. For example, with a threestage counter-current process, the volume of the process streams can bereduced by 35-50%. This greatly reduces the amount of water used in theprocess. The volume of process streams is also greatly reduced leadingto higher production throughput, short processing time and betterprocessing efficiency. In addition, the amount of water to be removedfrom the production process is also greatly reduced resulting in energysavings.

TABLE 1 The Results of Proximate Analysis on Defatted Microalgae Meal(Chlorella), Protein Concentrate and Hydrolyzed Protein Concentrate.Moisture Protein Ash Oil Crude Fibre Phytates Sample (%) (%, dwb) (%,dwb) (%, dwb) (%, dwb) (%, dwb) PDI Defatted Microalgae 10.9 54.8 33.61.65 5.68 0.46 29.3 (Chlorella) Protein 5.21 58.1 5.48 0.77 1.45 — —Concentrate

TABLE 2 Results of Proximate Analysis on Defatted Microalgae Meal(Chlorella), Protein Concentrate and Hydrolyzed Protein Concentrate.Moisture Protein Ash Oil Crude Fibre Carbohydrate Sample (%) (%, dwb)(%, dwb) (%, dwb) (%, dwb) (%, dwb) Defatted Microalgae 4.65 22.23 35.241.73 5.96 34.84 (Chlorella) Hydrolyzed Protein 2.86 58.88 12.15 2.142.94 23.89 Concentrate Microalgae Fiber 1.56 13.41 41.24 2.97 13.2129.17 Solids

1. A process for the production of a protein concentrate from macroalgaeand/or microalgae comprising: i) mixing the macroalgae and/or microalgaewith a first blending solvent to form a mixture; ii) optionally treatingthe mixture with phytase at a temperature and a pH suitable for phytaseactivity; iii) optionally adjusting the pH of the mixture to solubilizeproteins in the mixture; iv) subjecting the mixture to a g-forcesufficient to separate the mixture to form a) a fiber fraction, and b) aprotein fraction comprising (i) an insoluble protein fraction, and (ii)a soluble protein fraction; v) separating the fiber fraction from theprotein fraction and mixing the fiber fraction with a second blendingsolvent to form a fiber mixture; vi) treating the fiber mixture with aprotease at a temperature and a pH suitable for protease activity; vii)subjecting the fiber mixture to a g-force sufficient to separate thefiber mixture to form: a) a second fiber fraction, and b) a hydrolyzedprotein fraction, comprising (i) an insoluble protein fractioncomprising partially hydrolyzed and un-hydrolyzed protein, and (ii) asoluble hydrolyzed protein fraction; viii) optionally adjusting the pHof the protein fraction from step iv(b) to a pH suitable to precipitateproteins; x) separating the precipitated proteins from the proteinfraction; xi) optionally combining the precipitated proteins and thehydrolyzed protein fraction to form the protein concentrate.
 2. Theprocess according to claim 1, wherein the process further comprisesmixing the fiber fraction with the first blending solvent and repeatingstep iv) once, twice or three times.
 3. The process according to claim1, wherein the process further comprises mixing the second fiberfraction with the second blending solvent and repeating step vii) once,twice or three times.
 4. The process according to claim 1, wherein thefirst and second blending solvents comprise water, a saline solution ora polysaccharide solution.
 5. The process according to claim 4, whereinthe first and second blending solvents comprise water.
 6. The processaccording to claim 1, the ratio of the macroalgae and/or microalgae tothe first blending solvent is 1:3 to 1:30 (w/w) of algae to water. 7.The process according to claim 1, wherein the temperature suitable forphytase activity is between 20° and 60° C. and the pH suitable forphytase activity is between 2.0 and 7.0.
 8. The process according toclaim 1, wherein the temperature suitable for protease activity isbetween 30° and 70° C. and the pH suitable for protease activity isbetween 5.0 and 9.0.
 9. The process according to claim 1, wherein themixture and/or the fiber mixture is subjected to a g-force of between100 g and 500 g.
 10. The process according to claim 9, wherein themixture and/or the fiber mixture is subjected to a g-force of between150 g and 400 g.
 11. The process according to claim 10, wherein themixture and/or the fiber mixture is subjected to a g-force of between170 g and 350 g.
 12. The process according to claim 1, whereinseparating the mixture and/or the fiber mixture comprises using acentrifuge or a hydrocyclone.
 13. The process according to claim 1,wherein the pH suitable to precipitate the proteins in the proteinfraction is between 4.0 and 6.0.
 14. The process according to claim 1,further comprising the step of drying the protein concentrate to amoisture of between 4% and 8% (w/w).
 15. The process according to claim1, wherein the protein concentrate also comprises peptides and freeamino acids.
 16. The process according to claim 1, wherein the processis conducted as a counter-current process.
 17. A process for theproduction of a protein concentrate from macroalgae and/or microalgaecomprising: i) mixing the toasted oilseed meal with a first blendingsolvent to form a mixture; ii) optionally treating the mixture withphytase at a temperature and a pH suitable for phytase activity; iii)optionally adjusting the pH of the mixture to solubilize proteins in themixture; iv) subjecting the mixture to a g-force sufficient to separatethe mixture to form a) a fiber fraction, and b) a protein fractioncomprising (i) an insoluble protein fraction, and (ii) a soluble proteinfraction; v) separating the fiber fraction from the protein fraction andmixing the fiber fraction with a second blending solvent to form a fibermixture; vi) treating the fiber mixture with a protease at a temperatureand a pH suitable for protease activity; vii) subjecting the fibermixture to a g-force sufficient to separate the fiber mixture to form:a) a second fiber fraction, and b) a hydrolyzed protein fraction,comprising (i) an insoluble protein fraction comprising partiallyhydrolyzed and un-hydrolyzed protein, and (ii) a soluble hydrolyzedprotein fraction; viii) mixing the protein fraction from step iv(b) witha mixing solvent to precipitate proteins; ix) separating theprecipitated proteins from the protein fraction; and x) optionallycombining the precipitated proteins and the hydrolyzed protein fractionto form the protein concentrate.
 18. The process according to claim 17,wherein the process further comprises mixing the fiber fraction with thefirst blending solvent and repeating step iv) once, twice or threetimes.
 19. The process according to claim 17, wherein the processfurther comprises mixing the second fiber fraction with the secondblending solvent and repeating step vii) once, twice or three times. 20.The process according to claim 17, wherein the first and second blendingsolvents comprise water, a saline solution or a polysaccharide solution.21. The process according to claim 20, wherein the first and secondblending solvents comprise water.
 22. The process according to claim 17,the ratio of the macroalgae and/or microalgae to the first blendingsolvent is 1:3 to 1:30 (w/w) of algae to water.
 23. The processaccording to claim 17, wherein the temperature suitable for phytaseactivity is between 20° and 60° C. and the pH suitable for phytaseactivity is between 2.0 and 7.0.
 24. The process according to claim 17,wherein the temperature suitable for protease activity is between 30°and 70° C. and the pH suitable for protease activity is between 5.0 and9.0.
 25. The process according to claim 17, wherein the mixture issubjected to a g-force of between 100 g and 500 g.
 26. The processaccording to claim 25, wherein the mixture is subjected to a g-force ofbetween 150 g and 400 g.
 27. The process according to claim 26, whereinthe mixture is subjected to a g-force of between 170 g and 350 g. 28.The process according to claim 17, wherein separating the mixturecomprises using a centrifuge or a hydrocyclone.
 29. The processaccording to claim 17, wherein the mixing solvent comprises anethanol:water mixture, wherein the ethanol is present in an amountbetween 80% and 100% (v/v).
 30. The process according to claim 17,further comprising the step of drying the protein concentrate to amoisture of between 4% and 8% (w/w).
 31. The process according to claim17, wherein the protein concentrate also comprises peptides and aminoacids.
 32. The process according to claim 17, wherein the process isconducted as a counter-current process.
 33. A process for the productionof a protein isolate from a toasted oilseed meal comprising: i) mixingthe toasted oilseed meal with a first blending solvent to form amixture; ii) optionally treating the mixture with phytase at atemperature and a pH suitable for phytase activity; iii) optionallyadjusting the pH of the mixture to solubilize proteins in the mixture;iv) subjecting the mixture to a g-force sufficient to separate themixture to form a) a fiber fraction, and b) a protein fractioncomprising (i) an insoluble protein fraction, and (ii) a soluble proteinfraction; vi) separating the insoluble protein fraction from the solubleprotein fraction to recover therefrom an insoluble protein concentrateand a soluble protein extract; and vii) subjecting the soluble proteinextract to membrane filtration to recover therefrom the protein isolate.34. The process according to claim 33, wherein the process furthercomprises mixing the fiber fraction with the first blending solvent andrepeating step iv) once, twice or three times.
 35. The process accordingto claim 33, wherein the first blending solvent comprises water, asaline solution or a polysaccharide solution.
 36. The process accordingto claim 35, wherein the first blending solvent comprises water.
 37. Theprocess according to claim 33, the ratio of the macroalgae and/ormicroalgae to the first blending solvent is 1:3 to 1:30 (w/w) of algaeto water.
 38. The process according to claim 33, wherein the temperaturesuitable for phytase activity is between 20° and 60° C. and the pHsuitable for phytase activity is between 2.0 and 7.0.
 39. The processaccording to claim 33, wherein the mixture is subjected to a g-force ofbetween 100 g and 500 g.
 40. The process according to claim 39, whereinthe mixture is subjected to a g-force of between 150 g and 350 g. 41.The process according to claim 40, wherein the mixture is subjected to ag-force of between 170 g and 350 g.
 42. The process according to claim33, wherein separating the mixture comprises using a centrifuge or ahydrocyclone.
 43. The process according to claim 33, further comprisingthe step of drying the protein isolate to a moisture of between 4% and8% (w/w).
 44. The process according to claim 33, wherein the proteinisolate also comprises peptides and amino acids.
 45. The processaccording to claim 33, wherein the protein isolate is hydrolyzed toproduce peptides and free amino acids.
 46. The process according toclaim 33, wherein the process is conducted as a counter-current process.